Nonlinear microscopy, namely multiphoton imaging and second harmonic generation (SHG), is an established noninvasive technique useful for the imaging of extracellular matrix (ECM). Typically, measurements are performed in vivo on freshly excised tissues or biopsies. In this article, we describe the effect of rehydrating paraffin-embeddedsections on multiphoton and SHG emission signals and the acquisition of nonlinear images from hematoxylin and eosin (H&E)-stained sections before and after a destaining protocol. Our results reveal that bringing tissue sections to a physiological state yields a significant improvement in nonlinear signals, particularly in SHG. Additionally, the destaining of sections previously processed with H&E staining significantly improves their SHG emission signals during imaging, thereby allowing sufficient analysis of collagen in these sections. These results are important for researchers and pathologists to obtain additional information from paraffin-embedded tissues and archived samples to perform retrospective analysis of the ECM or gain additional information from rare samples. PMID:27018844

Interphase cytogenetic analysis using chromosome-specific probes is increasingly being used to detect chromosomal aberrations on paraffin-embedded tissue sections. However, quantitative analysis of the hybridization signal is confounded by the nuclear slicing that occurs during sectioning. To determine the sensitivity and accuracy of chromosome in situ hybridization for detecting numerical chromosomal aberrations on paraffin-embeddedsections, in situ hybridization was performed on sections derived from mixtures of cell populations with known frequencies of numerical chromosomal aberrations and the Chromosome Index (CI) was calculated (i.e., total number of signal spots/number of nuclei counted) as a quantitative measure of chromosome copy number. The presence of 25% or more monosomic or tetrasomic cells in a given population was easily detected as a change in CI (P < 0.05). Lower degrees of polysomy could be detected as a small percentage of nuclear fragments with > 2 signal spots. The CI was not significantly influenced by a change in section thickness from 4 to 8 microM, by an increase in cell size from 478 to 986 microM3, or by the choice of detection method (fluorescence vs. conventional bright-field microscopy). Comparative analysis of touch preparations and tissue sections from the corresponding breast tumors showed that CI accurately reflects the average copy number of chromosomes in intact nuclei and may actually be superior to in situ hybridization on whole nuclei for the detection of numerical chromosomal changes in defined histologic areas. This method is thus a sensitive and accurate means of studying genetic changes in premalignant and malignant tissue, and of assessing the genetic changes associated with specific phenotypes. PMID:7924678

The purpose of the present study was to evaluate the diagnostic accuracy of remote frozen sections examined by telepathology. The gold standard was the diagnosis made using direct examination of paraffin-embeddedsections. A consecutive series of 134 frozen-section cases were examined by six qualified pathologists. We used the Zeiss telepathology system with robot microscopy, which allowed different magnifications and fields of view to be chosen. The wide-area network used the TCP/IP protocol. The diagnosis made on the frozen sections was compared with the final diagnosis in the paraffin-embeddedsections. Times were recorded for each telepathology session, as well as the users comments on usability and software, and on any communication problems which occurred. In addition, we evaluated the importance of the macroscopic sampling of the surgical specimen, applied to each type of tissue. The diagnostic evaluation showed complete agreement in approximately 80% of cases, in 20% diagnosis was not possible due to insufficient quality of the slides. The median time for the telemedicine diagnosis was 14 min 30 sec.

The loss of antigenicity in archival formalin-fixed paraffin-embedded (FFPE) tissue sections negatively affects both diagnostic histopathology and advanced molecular studies. The mechanisms underlying antigenicity loss in FFPE tissues remain unclear. The authors hypothesize that water is a crucial contributor to protein degradation and decrement of immunoreactivity in FFPE tissues. To test their hypothesis, they examined fixation time, processing time, and humidity of storage environment on protein integrity and antigenicity by immunohistochemistry, Western blotting, and protein extraction. This study revealed that inadequate tissue processing, resulting in retention of endogenous water in tissue sections, results in antigen degradation. Exposure to high humidity during storage results in significant protein degradation and reduced immunoreactivity, and the effects of storage humidity are temperature dependent. Slides stored under vacuum with desiccant do not protect against the effects of residual water from inadequate tissue processing. These results support that the presence of water, both endogenously and exogenously, plays a central role in antigenicity loss. Optimal tissue processing is essential. The parameters of optimal storage of unstained slides remain to be defined, as they are directly affected by preanalytic variables. Nevertheless, minimization of exposure to water is required for antigen preservation in FFPE tissue sections. PMID:21411807

Superparamagnetic iron oxide particles are used as potent contrast agents in magnetic resonance imaging. In histology, these particles are frequently visualized by Prussian blue iron staining of aldehyde-fixed, paraffin-embedded tissues. Recently, zinc salt-based fixative was shown to preserve enzyme activity in paraffin-embedded tissues. In this study, we demonstrate that zinc fixation allows combining in situ zymography with fluorescence immunohistochemistry (IHC) and iron staining for advanced biologic investigation of iron oxide particle accumulation. Very small iron oxide particles, developed for magnetic resonance angiography, were applied intravenously to BALB/c nude mice. After 3 hours, spleens were explanted and subjected to zinc fixation and paraffinembedding. Cut tissue sections were further processed to in situ zymography, IHC, and Prussian blue staining procedures. The combination of in situ zymography as well as IHC with subsequent Prussian blue iron staining on zinc-fixed paraffin-embedded tissues resulted in excellent histologic images of enzyme activity, protease distribution, and iron oxide particle accumulation. The combination of all three stains on a single section allowed direct comparison with only moderate degradation of fluorescein isothiocyanate-labeled substrate. This protocol is useful for investigating the biologic environment of accumulating iron oxide particles, with excellent preservation of morphology. PMID:22954182

The use of digital fluorescence confocal microscopy in biological sciences has grown in recent decades due to the versatility of fluorescence imaging. The ability to selectively label specific morphological features, genetic mutations and/or chemical micro-environmental changes with discreet fluorescent labels allows a better understanding of the complex systems that regulate cellular processes. Specimens can range in size from single cells to tissue sections and tissue arrays, which can occupy the entire surface of a microscope slide (25mm x 70mm). Using a confocal scanning laser MACROscope, a wide-area confocal imaging system (Biomedical Photometrics Inc.), it is possible to image these large specimens at high resolution, without the need to tile many small microscope fields. A hyperspectral imaging (HSI) mode has been added to the MACROscope system to assess the use of HSI in the removal/separation of tissue autofluorescence from digital images of fluorescently-labeled paraffin-embedded, formalin-fixed tissue sections. In pathology and immunohistochemistry applications this autofluorescence can hinder, or even prevent, detection of the applied fluorescent label(s). In the present study, fluorescence emission from the specimen was sampled at ~7 nm bandwidths across 32 channels, amounting to viewing ~220 nm of the visible spectrum as a hyperspectral data cube. The data cube was then processed to remove the contributions from autofluorescence, leaving only the signal from the fluorophore(s) of interest. Comparisons are drawn from HSI obtained with a commercial hyperspectral confocal microscope (Zeiss LSM 510 META) employing image tiling. The initial results demonstrate the ability to spectrally unmix the tissue autofluorescence in large tissue sections.

Thirty-nine skin tumors of epithelial, mesenchymal, and neuroectodermal origin were studied using antibodies against intermediate filaments and other cell proteins. Formol-fixed and paraffin-embedded material was reconstituted and stained with antibodies against epithelial cells (keratin, epithelial membrane antigen, carcinoembryonic antigen), mesenchymal and histiocytic cells (vimentin, alpha-1-antichymotrypsin, alpha-1-antitrypsin, lysozyme), nerve tissue (neurofilament, glial fibrillary acidic protein, myelin basic protein, myelin-associated protein, neuron-specific enolase), vessels (factor-VIII-related protein), basal cell lamina (laminin) and S-100 protein. Tumor cells displayed the same antibody pattern found in the normal cell type. It is recommended that immunotyping be started with three antibodies to allow gross classification into epithelial (keratin positive), mesenchymal (vimentin positive) and neuroectodermal (vimentin and S-100 protein positive) tumors; then, in a second step, the tumors can be subclassified by the other more specific antibodies listed above. All antibodies used in this study are commercially available and provide reliable results. PMID:3553072

Fluorescence in situ hybridization has become a major tool for analysis of gene and chromosome copy number in normal and malignant tissue. The technique has been applied widely to fresh tissue and dispersed formalin-fixed, paraffin-embedded archival tissue, but its use on sections of archival tissue has largely been limited to sections < 6 mu thick. This does not provide intact, uncut nuclei for accurate analysis of gene or chromosome copy number. We report here a method of hybridization to sections > 20 microns thick that overcomes these difficulties. Key developments were the use of DNA probes directly labeled with fluorochromes and optical sectioning using laser-scanning confocal microscopy. Images Figure 2 Figure 3 PMID:8311111

AIMS: To determine whether immunohistochemistry applied to paraffin wax embedded biopsy tissue can be used to distinguish between B-small lymphocytic lymphoma (B-SLL) and mantle cell lymphoma (MCL). METHODS: Formalin fixed, paraffin wax embedded tissue blocks of 12 cases of B-SLL and 12 cases of MCL were retrieved from the files of the Department of Pathology, Southampton University Hospitals Trust. Following antigen retrieval, where appropriate, sections were stained for CD3, CD5, CD20, CD23, CD43, Cyclin D, PGP9.5, and MIB1 using a streptavidin-biotin complex technique. RESULTS: CD20 stained the neoplastic cells of B-SLL and MCL, and CD3 labelled the reactive T cells in these tumours. In B-SLL, the T cells were generally dispersed among the tumour cells, whereas in MCL they often formed bands around tumour cell nodules. CD5 could be detected on T cells, following antigen retrieval. The level of expression on B cells of B-SLL and MCL was generally too low to allow detection in paraffin wax embedded tissues. CD23 stained B-SLL but not MCL. However, it could be detected in only five of the 12 cases of B-SLL. CD43 could be detected in most cases of B-SLL and MCL. It is not, therefore, of value in distinguishing between these tumours. It will, however, help in the differentiation of B-SLL and MCL from other low grade B cell lymphomas, such as follicle centre cell and marginal zone lymphomas. Cyclin D was expressed in all of the MCL but in none of the B-SLL. PGP9.5 showed reactivity in most cases of MCL and much weaker reactivity in B-SLL. The proliferation indexes of MCL were generally higher than those of B-SLL, as measured by MIB1 labelling. Both tumours, however, showed a wide range of values and considerable overlap. CONCLUSION: Staining for Cyclin D is the most reliable immunohistochemical mean of differentiating between B-SLL an MCL. High levels of PGP9.5, expressed in MCL, may be related to the degradation of Cyclin D by the ubiquitin pathway. Images PMID

MALDI-imaging MS (IMS) with MSMS analysis is a new powerful tool for the identification of not only disease-related proteins in formalin-fixed paraffin-embedded (FFPE) tissue sections but also protein/peptides/drugs/medicine in fresh-frozen tissues. IMS is used to reveal the mass profiles and spatial distribution of proteins in tissue sections and/or digested peptides derived from deposited protein in pathologic organs and then MSMS analysis identifies the amino acid sequence of the detected proteins in the tissue section. Moreover, on-tissue digestion combined with the MALDI-IM-TOF-IMS approach allows a proteomics "bottom-up" strategy with clinical samples, especially perioperative isolated tissues and FFPE tissues conserved for a long time in a clinical sample bank. The mass barcode-like image (MBI) on a longitudinal sliced hair by IMS is used in the selected reaction monitoring mode for serially chronological monitoring and traceability every few hours after drug and medicine intake. The advances of quantitative MBI for sliced sections of hair allow a new universal standardized assessment of drugs and medicines throughout the drug history. PMID:22568093

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embeddedsections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes. Images PMID:9536283

We report new methods for multilabel immunofluorescence (MIF) and reprobing of antigen epitopes on the same formalin-fixed paraffin-embedded (FFPE) sections. The MIF method includes an antigen-retrieval step followed by multilabel immunostaining and examination by confocal microscopy. As examples, we illustrate epitopes localized to the apical and basolateral membranes, and the cytoplasm of enterocytes of normal small intestine and in cases of congenital enteropathies (microvillous inclusion disease and congenital tufting enteropathy). We also demonstrate localization of the bile salt excretion pump protein (BSEP) in bile canalicular membrane of normal hepatocytes and in cases of primary sclerosing cholangitis. To demonstrate colocalization of cytoplasmic and nuclear epitopes we analyzed normal control and hyperplastic pulmonary neuroendocrine cells (PNEC) and neuroepithelial bodies (NEBs), presumed airway sensors in the lungs of infants with bronchopulmonary dysplasia (BPD). As cytoplasmic markers we used anti-bombesin or anti-synaptic vesicle protein 2 (SV2) antibody, respectively, and for nuclear localization, antibodies against neurogenic genes mammalian achaete-scute homolog (Mash1) and prospero homeobox 1 (Prox1), essential for NEB cells differentiation and maturation, hypoxia-inducible factor 1α (HIF1α) a downstream modulator of hypoxia response and a proliferation marker Ki67. The reprobing method consisted of removal of the previously immunolabeled target and immunostaining with different antibodies, facilitating colocalization of enterocyte brush border epitopes as well as HIF1α, Mash1 and Prox1 in PNEC/NEB PNEC and NEBs. As these methods are suitable for routine FFPE pathology samples from various tissues, allowing visualization of multiple epitopes in the same cells/sections with superior contrast and resolution, they are suitable for a wide range of applications in diagnostic pathology and may be particularly well suited for precision medicine

The use of tissue softeners to enhance the quality of tissue sections of heavily keratotic tissue is not widely published. There are very few indicators in the scientific literature that attempt to compare and contrast the benefits and disadvantages of such techniques, as most are passed down through word of mouth rather than through published data. This study attempts to present a preliminary evaluation of several methods employing tissue softeners to facilitate the preparation of reproducible, good-quality formalin-fixed, paraffin-embeddedsections of nail tissue. A standard 10-minute surface application of each softener is employed for all paraffin-embedded tissue in order to ensure consistency. The results show that the use of Veet (hair remover), Fairy Liquid or fabric conditioner provides the most beneficial results. Thus, widely available products can be used in preference to specific commercially produced reagents that have no clear benefits and can cost considerably more to purchase. This study will form the basis of a more in-depth evaluation of the most beneficial softeners, in an attempt to determine optimal parameters for their use in routine histopathology laboratories. PMID:19055107

Antigen retrieval is an immunohistochemical procedure that results in better exposure of target antigens in aldehyde-fixed, paraffin-embedded tissue sections to antibodies. However, the commercially recommended or conventional protocols for antigen retrieval do not always succeed in expressing the target antigen. Here, an improved method was developed for antigen retrieval from aldehyde-fixed, paraffin-embedded histological sections. Proliferating cell nuclear antigen (PCNA), tight junction proteins Claudin-2 and Claudin-7, and water channel aquaporins in kidney tissue were selected as test antigens. Typically, PCNA and Claudin-2 and Claudin-7 show negative, weak, or nonspecific immunoreactions with conventional antigen retrieval methods using microwave heating. In the present study, microwave heating was performed twice with an interval of 30 min between the two steps to allow the buffer solution to cool. Sodium citrate buffer (10 mM sodium citrate, pH 6.0) was used for PCNA, and Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 9.0) was used for the Claudins. Compared with conventionally prepared tissues, the tissues exhibited both enhanced and specific immunostaining, and well-preserved morphology. In conclusion, the conventional protocol could be supplemented with a second microwave heating step to improve the expression of antigens that do not respond well to the conventional method. PMID:27002723

Two methods for colorimetric in situ DNA probe hybridization (CISH) assays on paraffin-embedded tissue sections were compared. The heated method used heat (90-100°C) to denature DNA in the sample prior to probe hybridization, while the unheated method used a standard hybridization temperature of 42°C. Both procedures were tested on tissue samples that harbored the mollusk protozoan pathogens Perkinsus marinus, P. chesapeaki, or Haplosporidium nelsoni, the protozoan and bacterial fish pathogens Myxobolus cerebralis (myxosporidean) or Renibacterium salmoninarum (bacterial), or the crab viral pathogen Callinectes sapidus reovirus. Samples were fixed in either formalin or Davidson's fixative and embedded in paraffin for histological examination. The heated method is labor intensive and highly prone to human error, while the unheated method is less labor intensive and can be completed in a shorter period of time. Both methods yielded similar hybridization results. The use of complex and expensive prehybridization buffers did not improve the performances of the tested CISH assays. Prehybridization heat denaturation of DNA in assayed samples increased both assay duration and loss of samples but did not improve hybridization signals. PMID:23697605

Immunohistochemistry (IHC) is an efficient technique to detect cellular localizations of the proteins in paraffin-embedded tissues. It allows specific proteins to be visualized by the interaction of antibodies with an enzyme-substrate-chromogen system. Here, we describe indirect immunohistochemistry method for paraffin-embedded mouse ovaries fixed with Bouin's Fixative. PMID:27557588

Four doves (Nos. 1–4 birds) affected with neurological signs (ataxia, circling and torticollis) were investigated pathologically and microbiologically. Viral isolation was tried from the tracheal and cloacal swabs of all 4 birds and from liver, spleen, kidney, heart, lung and brain of Nos. 1 and 2 birds. No viruses were isolated from 4 birds, but they had high serum antibody titers against avian paramyxovirus 1 (APMV-1). Histologically, they had the characteristic histological changes of pigeon APMV-1 infection; nonpurulent encephalitis and interstitial nephritis. Immununohistochemically, APMV-1 antigens were detected in the necrotic renal tubular epithelial cells of 1 bird of them (No. 3 bird). Detection of APMV-1 ribonucleic acid (RNA) from formalin-fixed and paraffin-embedded (FFPE) sections was attempted by reverse transcription-polymerase chain reaction (RT-PCR). Sequencing the RT-PCR product showed the virus RNA belonged to the same APMV-1 genotype (VI) as the strains isolated from the world previous cases of pigeon APMV-1 infection. The RT-PCR of FFPE sections and sequencing of RT-PCR products are useful for molecular epidemiology of the virus when viral isolation from fresh samples is unsuccessful. PMID:25816803

Four doves (Nos. 1-4 birds) affected with neurological signs (ataxia, circling and torticollis) were investigated pathologically and microbiologically. Viral isolation was tried from the tracheal and cloacal swabs of all 4 birds and from liver, spleen, kidney, heart, lung and brain of Nos. 1 and 2 birds. No viruses were isolated from 4 birds, but they had high serum antibody titers against avian paramyxovirus 1 (APMV-1). Histologically, they had the characteristic histological changes of pigeon APMV-1 infection; nonpurulent encephalitis and interstitial nephritis. Immununohistochemically, APMV-1 antigens were detected in the necrotic renal tubular epithelial cells of 1 bird of them (No. 3 bird). Detection of APMV-1 ribonucleic acid (RNA) from formalin-fixed and paraffin-embedded (FFPE) sections was attempted by reverse transcription-polymerase chain reaction (RT-PCR). Sequencing the RT-PCR product showed the virus RNA belonged to the same APMV-1 genotype (VI) as the strains isolated from the world previous cases of pigeon APMV-1 infection. The RT-PCR of FFPE sections and sequencing of RT-PCR products are useful for molecular epidemiology of the virus when viral isolation from fresh samples is unsuccessful. PMID:25816803

The present report describes a real-time PCR-based procedure to reliably determine the quantity of Leishmania amastigotes in relation to the amount of host tissue in histological skin sections from canine and equine cases of cutaneous leishmaniasis. The novel diagnostic Leishmania-PCR has a detection limit of <0.02 amastigotes per μg tissue, which corresponds well to the detection limit of immunohistochemistry and is far beyond that of conventional histology. Our results emphasise the importance of PCR to complement routine histology of cutaneous leishmaniasis cases, particularly in laboratories in which no immunohistochemical assay is available. PMID:26427730

A multimodal mass spectrometry imaging (MSI) based approach was used to characterize the molecular content of crystal-like structures in a frozen and paraffinembedded piece of a formalin-fixed rabbit kidney. Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) imaging and desorption electrospray ionization (DESI) mass spectrometry imaging were combined to analyze the frozen and paraffinembedded sample without further preparation steps to remove the paraffin. The investigated rabbit kidney was part of a study on a drug compound in development, in which severe renal toxicity was observed in dosed rabbits. Histological examination of the kidney showed tubular degeneration with precipitation of crystal-like structures in the cortex, which were assumed to cause the renal toxicity. The MS imaging approach was used to find out whether the crystal-like structures were composed of the drug compound, metabolites, or an endogenous compound as a reaction to the drug administration. The generated MALDI-MSI data were analyzed using principal component analysis. In combination with the MS/MS results, this way of data processing demonstrates that the crystal structures were mainly composed of metabolites and relatively little parent drug.

Epidermal growth factor receptor (EGFR) has been the subject of much research since it was first described as a prognostic factor in breast cancer. The assay methods used and results obtained vary widely between studies. In this study 88 primary breast cancers were assayed for EGFR using a novel immunohistochemical assay performed on paraffin-embeddedsections. The monoclonal antibody used was raised against purified, denatured EGFR, reacts with an epitope on the external domain and does not interfere with ligand binding. Twenty-two per cent of the tumours were EGFR positive using this assay. The results obtained were significantly correlated with those obtained by ligand-binding assay (r = 0.621, P = 0.011). The concordance rate was 82% (P < 0.001). The majority of discordant results could be explained by the presence of benign breast tissue and other non-malignant elements which could be seen to express EGFR on the immunohistochemical assay and were excluded from the score for this, but would be incorporated into ligand-binding assay results. The well-established inverse relationship between EGFR (as measured by this assay) and oestrogen receptor (ER) was seen (chi 2 = 24.9, P < 0.0001). In addition, in this exploratory study on a limited tumour set, EGFR was a significant adverse prognostic factor (on univariate but not multivariate analysis) for both relapse-free survival (P = 0.02) and overall survival (P = 0.03) when measured by this immunohistochemical assay, but was not significant when measured by ligand-binding assay. Images Figure 1 Figure 2 PMID:7779717

Estrogen receptors (ER) were independently analyzed using dextran-coated charcoal assays (ER-DCC) and immunohistochemical assays in frozen (ER-ICA) and paraffin-embedded tissue (ER-PAR) from 130 human breast cancer specimens drawn from postmenopausal high-risk patients registered in the Danish Breast Cancer Cooperative Group. ER was best detected with the ER-DCC assay followed by the ER-ICA (relative sensitivity 87%) and the ER-PAR assays (relative sensitivity 71%). The semiquantified staining features of the immunohistochemical assays were statistically significantly correlated with each other and with ER-DCC. Analysis of disease-free interval (DFI) and overall survival (OS) showed that all assays allowed statistically significant discrimination between a high risk and a low risk group, although the sensitivity differences tended to be reflected as small differences in clinical discriminatory power. The patient groups were then stratified according to adjuvant treatment [radiotherapy (RT) versus radiotherapy and tamoxifen (RT + TAM)]. The survival advantage was tied primarily to the receptor status itself in the steroid-binding assays, but was linked to both the receptor status and the adjuvant treatment in the immunohistochemical assays. Thus, the relative risks in terms of DFI and OS were of the same relative magnitude in the RT and RT + TAM groups for ER-DCC assays using a cut-off level of 10 fmol/mg cytosol protein, while there were large differences in the relative risks between RT and RT + TAM groups for ER-ICA and ER-PAR assays. We conclude that an ER assay in fresh tissue should be given first priority, but if there is no fresh tissue, an ER assay in paraffin-embedded tissue offers a reasonably good alternative as a prognosticator and an equivalent alternative as a predictor of the response to endocrine treatment. PMID:1694085

The refractive indices, absorption coefficients, and complex dielectric constants of paraffin-embedded brain glioma and normal brain tissues have been measured by a terahertz time-domain spectroscopy (THz-TDS) system in the 0.2- to 2.0-THz range. The spectral differences between gliomas and normal brain tissues were obtained. Compared with normal brain tissue, our results indicate that paraffin-embedded brain gliomas have a higher refractive index, absorption coefficient, and dielectric constant. Based on these results, the best THz frequencies for different methods of paraffin-embedded brain glioma imaging, such as intensity imaging, coherent imaging with continuum THz sources, and THz pulsed imaging with short-pulsed THz sources, are analyzed.

A method for the detection of Salmonella based on fluorescence in situ hybridization (FISH) has been developed and applied for the direct detection of Salmonella in pure cultures and in formalin-fixed, paraffin-embedded tissue sections. On the basis of the 23S rRNA gene sequences representing all of the S. enterica subspecies and S. bongori, an 18-mer oligonucleotide probe was selected. The specificity of the probe was tested by in situ hybridization to bacterial cell smears of pure cultures. Forty-nine of 55 tested Salmonella serovars belonging to subspecies I, II, IIIb, IV, and VI hybridized with the probe. The probe did not hybridize to serovars from subspecies IIIa (S. arizonae) or to S. bongori. No cross-reaction to 64 other strains of the family Enterobacteriaceae or 18 other bacterial strains outside this family was observed. The probe was tested with sections of formalin-fixed, paraffin-embedded tissue from experimentally infected mice or from animals with a history of clinical salmonellosis. In these tissue sections the probe hybridized specifically to Salmonella serovars, allowing for the detection of single bacterial cells. The development of a fluorescence-labelled specific oligonucleotide probe makes the FISH technique a promising tool for the rapid identification of S. enterica in bacterial smears, as well as for the detection of S. enterica in histological tissue sections. PMID:9316923

Angioimmunoblastic T-cell lymphoma (AITL) is an infrequent subtype of peripheral T-cell lymphoma derived from follicular helper T cells. Recently, a somatic G17V RHOA gene mutation has been reported. In this article, we examined the RHOA G17V mutation in 18 cases of AITL by 3 different techniques of Sanger sequencing, fully automated SNP genotyping, and deep sequencing, using routine diagnostic formalin-fixed paraffin-embedded tissue. The RHOA G17V mutation was detected in 10 cases (56%). Among the 10 mutated cases, 8 cases were detected by all 3 methods. The status of RHOA mutation was subsequently compared with the clinicopathologic characteristics of AITL. RHOA-mutated AITL (10 cases) was clinically characterized by high serum IL-2R and a poor ECOG performance status. By immunohistochemistry, expression of CD10, PD-1, CXCL13, and CCR4 and a wide distribution of CD21(+) follicular dendritic cells were observed in RHOA-mutated cases. Among these, CCR4 expression and the CD21(+) network in RHOA-mutated AITL cases were more extensive than in the RHOA mutation-negative AITL cases (P<0.05). Thus, RHOA-mutated AITL cases are more characteristic of follicular helper T cells, and the presence of such a mutation is an important marker for AITL. PMID:27158755

This work seeks to obtain the properties of paraffin-embedded breast cancer tumor tissues using transmission imaging and spectroscopy. Formalin-fixed and paraffin-embedded breast tumors are first sectioned into slices of 20 μm and 30 μm and placed between two tsurupica slides. The slides are then scanned in a pulsed terahertz system using transmission imaging. The tissue regions in adjacent pathology section are compared to the transmission imaging scan in order to define a region of points over which to average the electrical properties results from the scan.

The present study was designed to evaluate the efficacy of different microwave pretreatment methods to retrieve microtubule-associated protein 2 (MAP-2) immunoreactivity in formalin-fixed, paraffin-embedded guinea pig brain sections. Brain sections, microwave pretreated in boiling sodium citrate, citric acid, Tris hydrochloride, and EDTA buffers of pH 4, 6, and 8, were labeled with four different clones of MAP-2 monoclonal antibodies. No MAP-2 immunoreactivity was observed in control sections processed without microwave pretreatment. Optimal MAP-2 immunoreactivity was observed only when MAP-2 antibody clone AP18 was used in conjunction with citric acid buffer of pH 6.0. Using this combination, brain sections from nerve agent soman-exposed guinea pigs were found to exhibit marked reduction in MAP-2 immunostaining in the hippocampus. These observations suggest that the clone of the antibody in addition to the type and pH of antigen retrieval (AR) solution are important variables to be considered for establishing an optimal AR technique. When studying counterpart antigens of species other than that to which the antibodies were originally raised, different antibody clones must be tested in combination with different microwave-assisted AR (MAR) methods. This MAR method makes it possible to conduct retrospective studies on archival guinea pig brain paraffin blocks to evaluate changes in neuronal MAP-2 expression as a consequence of chemical warfare nerve agent toxicity. PMID:15817147

In this book chapter we report our own experience of mutational analysis in selecting tailored anticancer treatments for solid tumors. Our Department of Advanced Biotechnologies and Bioimaging, IRCCS San Raffaele Pisana, Rome, Italy, routinely performs pharmacogenetic screenings for different genes such as K-ras, BRAF, KIT, PDGFRα, and EGFR on paraffin-embedded cancer sections. Therefore, the chapter describes the mutational analysis procedures on paraffin-embedded tumors aimed to predict individual response to anticancer therapy. These molecular diagnostic methodologies may help us in improving the translational impact of genetic information on clinical practice. PMID:25150866

A single paraffin-embedded liver section was submitted from a research flock of Japanese quail that had revealed focal infiltrations of immature lymphocytes within multiple visceral organs. Tumor cells were characterized as T-cells positive for Marek's disease virus (MDV) pp38 antigen by IHC dual st...

RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tissue is commonly evaluated in both investigative and diagnostic pathology. However, the quality of the data is directly impacted by RNA quality. The RNA integrity number (RIN), an algorithm based on a combination of electrophoretic features, is widely applied to RNA isolated from paraffin-embedded tissue, but it is a poor indicator of the quality of that RNA. Here we describe the novel paraffin-embedded RNA metric (PERM) for quantifying the quality of RNA from FFPE tissue. The PERM is based on a formula that approximates a weighted area-under-the-curve analysis of an electropherogram of the extracted RNA. Using biochemically degraded RNAs prepared from experimentally fixed mouse kidney specimens, we demonstrate that PERM values correlate with mRNA transcript measurements determined using the QuantiGene system. Furthermore, PERM values correlate with real-time PCR data. Our results demonstrate that the PERM can be used to qualify RNA for different end-point studies and may be a valuable tool for molecular studies using RNA extracted from FFPE tissue. PMID:27177816

The refractive indices, absorption coefficients and complex dielectric constants spectra of paraffin-embedded brain glioma and normal brain tissues have been measured by a terahertz time domain spectroscopy (THz-TDS) system in the range of 0.2 - 2.0 THz. The spectral differences between glioma and normal brain tissues were obtained. Our results indicate that, compared with normal tissue, glioma had higher refractive index, absorption coefficient, and dielectric constant. Based on these results, the suitable frequency components for different methods of glioma imaging (intensity imaging, coherent imaging and terahertz pulsed imaging) are analyzed.

In general, it is believed that the extraction of proteins from formalin-fixed paraffinembedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools. PMID:18228195

Adipocyte size is a major modulator of endocrine functioning of adipose tissue and methods allowing accurate determination of adipocyte size are important to study energy metabolism. The aim of this study was to assess the relative shrinkage of adipocytes before and after weight loss by comparing adipose tissue from the same subjects embedded in paraffin and plastic. 18 healthy subjects (5 males and 13 females) aged 20-50 y with a BMI of 28-38 kg/m² followed a very low energy diet for 8 weeks. Adipose tissue biopsies were taken prior to and after weight loss and were processed for paraffin and plastic sections. Parameters of adipocyte size were determined with computer image analysis. Mean adipocyte size was smaller in paraffin compared to plastic embedded tissue both before (66 ± 4 vs. 103 ± 5 μm, P < 0.001) as after weight loss (62 ± 4 vs. 91 ± 5 μm, P < 0.001). Relative shrinkage of adipocytes in paraffinembedded tissue in proportion to plastic embedded tissue was not significantly different before and after weight loss (73 and 69%, respectively). Shrinkage due to the type of embedding of the adipose tissue can be ignored when comparing before and after weight loss. Plastic embedding of adipose tissue provides more accurate and sensitive results. PMID:24331678

Multiplexed miRNA fluorescence in situ hybridization (miRNA FISH) is an advanced method for visualizing differentially expressed miRNAs, together with other reference RNAs, in archival tissues. Some miRNAs are excellent disease biomarkers due to their abundance and cell-type specificity. However, these short RNA molecules are difficult to visualize due to loss by diffusion, probe mishybridization, and signal detection and signal amplification issues. Here, we describe a reliable and adjustable method for visualizing and normalizing miRNA signals in formalin-fixed paraffin-embedded (FFPE) tissue sections. PMID:25218385

A histological study was carried out on 58 formalin-fixed and paraffin-embedded samples of placenta from sheep and goats that had aborted, and the placental lesions were graded. Sequential histological sections of each cotyledon were then immunostained with specific antibodies and used for PCR detection of Chlamydophila abortus, Coxiella burnetii, Salmonella Abortusovis, Brucella melitensis, Listeria monocytogenes and Toxoplasma gondii. Most of the cotyledons showed different degrees of placentitis. The proportional agreement between the two techniques was 0.879 (kappa value 0.746). C abortus was the most prevalent pathogen. Mixed infections were common. PMID:19666916

This study aimed to deparaffinize formalin-fixed paraffin-embedded (FFPE) tissues using hot water instead of xylene and measuring the quantity and quality of the extracted DNA from the respective tissues. To deparaffinize the tissue sections with hot water, small sections were exposed to 90 °C distilled sterile water. After 25 FFPE tissue samples were deparaffinized with the hot water method, DNA was then extracted. The mean of optical density and the ratio of absorbance of the DNA solution were 220.01 ± 36.1 ng/μl and 1.65 ± 0.1, respectively. Polymerase chain reaction (PCR) analysis of the toll-like receptor 4(TLR4) gene showed that the method can be used as a tool for different applications. PMID:27287960

Described here is an immunohistochemical technique using the commercially available monoclonal progesterone receptor (PR) antibody KD 68 in routinely fixed and paraffin-embedded breast carcinomas and lymph node metastases. The authors' technique is compared with several incubation variations. The method applying the primary antibody in a dilution of 1:10 overnight followed by a biotinylated second antibody showed the best results when Triton X-100 was added to the buffer. Using this method, comparison with the results on frozen sections of 34 breast carcinomas yielded a significant concordance of 94%. Correlation between the results on paraffinsections and those obtained by the standard dextran-coated charcoal cytosol assay was 80%. The value of the method for predicting endocrine therapy response was shown in 20 patients. Thus the reliability of the method has been demonstrated and was applied on 151 lymph node metastases and the corresponding primary breast carcinomas from 50 patients. Generally PR content in the metastases was lower than in the primary tumors (p < 0.001). This finding indicates that evaluation of PR in lymph node metastases should be included in the decision for endocrine therapy of breast cancer. PMID:7686056

Background: DNA extraction from paraffin wax embedded tissue requires special protocols, and most described methods report an amplification success rate of 60–80%. Aims: To propose a simple and inexpensive protocol consisting of xylene/ethanol dewaxing, followed by a kit based extraction. Method: Xylene/ethanol dewaxing was followed by a long rehydration step and a kit based DNA extraction step. Results: This method produced a 100% amplification success rate for fragments of 121 to 227 bp for tamponated formalin fixed paraffin wax embedded tissue. Conclusion: This cost effective and non-laborious protocol can successfully extract DNA from tamponated formalin fixed paraffin wax embedded tissue and should facilitate the molecular analysis of a large number of archival specimens in retrospective studies. PMID:16049299

Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ(2) (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ(2) data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraffin-embedded mouse lung tissue fixed in different fixatives. PMID:24722432

The molecular investigation of archived formalin-fixed, paraffin-embedded (FFPE) tissue samples provides the chance to obtain molecular patterns as indicatives for treatment and clinical end points. MALDI mass spectrometry imaging is capable of localizing molecules like proteins and peptides in tissue sections and became a favorite platform for the targeted and non-targeted approaches, especially in clinical investigations for biomarker research. In FFPE tissues the recovery of proteomic information is constrained by fixation-induced cross-links of proteins. The promising new insights obtained from FFPE in combination with the comprehensive patients' data caused much progress in the optimization of MS imaging protocols to investigate FFPE samples. This review presents the past and current research in MALDI MS imaging of FFPE tissues, demonstrating the improvement of analyses, their actual limitations, but also the promising future perspectives for histopathological and tissue-based research. PMID:24838644

In the present study, dehydrated human colon tissues embedded in paraffin were studied at THz frequency. A compact THz imaging system with high numerical aperture optics was developed for the analysis of adenocarcinoma-affected colon sections, in transmission and reflection geometry. A comprehensive analysis of the THz images revealed a contrast up to 23% between the neoplastic and control tissues. Absorption and reflection THz images demonstrated the possibility to distinguish adenocarcinoma-affected areas even without water in the tissue, as the main contrast mechanism in THz measurements has been observed to be water absorption in in vivo or freshly excised tissues. The present results corroborate with previous histologic findings in the same tissues, and confirm that the contrast prevails even in dehydrated tissues.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies. PMID:8396155

Introduction: Formalin fixed paraffinembedded tissue are regularly employed in TSE diagnosis by IHC, the standard by which all other diagnostic protocols are currently judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot...

Formalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically...

Background Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffinembedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data. Methods FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging. Results The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections. Conclusion We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis. PMID:26030902

Formalin-fixed, paraffin-embedded (FFPE) sections of breast tissue are used by pathologists to correctly type and grade the primary tumor and to assess the extent of a patient's disease. The cut sections represent a reproducible likeness of the morphology of the tissue when viewed through a microscope, although the fixation technique creates some artifacts. What is not known is how the sections differ chemically from how the tumor would look or behave within the breast. Raman spectroscopy is, like many other optical techniques, fast, noninvasive, and generally inexpensive. The advantage Raman has over other techniques is its powerful ability to identify specific chemicals, molecules, and bonds within a sample. Using Raman spectroscopy the chemicals present in both fresh tissue and FFPE sections can be identified and compared, allowing any differences between them to be identified. This information may be useful to the pathologist as an aid to further treatment regimes or novel molecular techniques, and as an aid to patient management. If these sections are found to be chemically similar to fresh tissue, they could be used to further characterize breast tumors, particularly rare tumors, using Raman spectroscopy.

Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin-fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin-fixed paraffin-embedded tissue. PMID:26708177

Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue. PMID:26708177

A method is described for the preparation of monolayer smears from paraffin-embedded tissue. The smears are suitable for automated image analysis and DNA measurements while still allowing interpretation of nuclear morphology. The proposed technique uses enzyme treatment and syringing for cell dispersal. The preparation of cell monolayers is performed by cytocentrifugation. After staining the specimens with gallocyanin, nuclear DNA can be measured. Automated DNA measurements using the Leyden Television Analysis System (LEYTAS) showed coefficients of variation of 4.5% for the diploid cell population of suspended benign tissue. After DNA measurements, the specimens are counterstained using orange G and eosin. Since gallocyanin has spectral properties similar to those of hematoxylin, the obtained end product is comparable to specimens stained according to the routinely used Papanicolaou procedure. Using this technique, image cytometry can be applied to paraffin-embedded tissue in combination with conventional cytomorphologic study of the cells. PMID:3304329

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries. PMID:25229221

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h. PMID:17653827

New technologies allow for genome-scale measurement of DNA methylation. In an effort to increase the clinical utility of DNA methylation as a biomarker, we have adapted a commercial bisulfite epigenotyping assay for genome-wide methylation profiling in archival formalin-fixed paraffin-embedded pathology specimens. This chapter takes the reader step by step through a biomarker discovery experiment to identify phenotype-correlated DNA methylation signatures in routine pathology specimens. PMID:22081342

A simple and efficient method of DNA extraction from paraffin wax embedded tissues using a spin cartridge system is described. Such DNAs were shown to be suitable for amplification by the polymerase chain reaction, which targeted two human papillomavirus genes and one globin fragment giving rise to products of 450, 150, and 110 base pairs, respectively. Different human tissues, stored for up to 20 years, were successfully amplified, demonstrating the usefulness of this very simple procedure for retrospective studies. PMID:9624421

A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post-inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR-based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin-embedded tissues. PMID:24274927

In a study on 26 patients with autoimmune diseases the immunostaining for immunoglobulins and complement in frozen sections was compared with that in formalin-fixed and paraffin-embeddedsections. The formalin-fixed material of pemphigus vulgaris, lupus erythematosus, bullous pemphigoid, and dermatitis herpetiformis (Duhring's disease) was reconstituted and stained by means of the standard two-step technique (TST), the peroxidase-antiperoxidase technique (PAP), and the streptavidin-biotin method (SAB). In comparison with frozen sections, immunoglobulins and complement could also be demonstrated in formalin-fixed sections in all cases of pemphigus vulgaris and in 85% of cases of discoid lupus erythematosus, but in only 60% of cases of bullous pemphigoid or Duhring's disease. PAP and SAB proved to be about equally reliable, but TST was significantly less dependable. PMID:3403272

Aim—To analyse the effect of sectioning on the assessment of karyotypic heterogeneity by interphase cytogenetics in paraffin wax embedded normal squamous epithelium and to apply the principles derived to invasive cervical carcinoma. Methods—Normal male (n = 5) and female (n = 5) squamous epithelia were hybridised with peri-centromeric repeat probes specific for chromosomes X (DXZ1) and 17 (D17Z1) individually and in combination to assess the effect of sectioning on mono-, di-, tri-, and tetrasomic populations. Section thickness, interobserver variation and variation between different areas of the epithelium were evaluated. Invasive squamous carcinomas of the cervix (n = 5) were then hybridised with the DXZ1 probe and intratumoral heterogeneity was assessed by comparison of signal distributions obtained from different areas. Results—The optimum section thickness for the assessment of normal epithelium was 6 μm. Variation in the expected signal number in the range 1-4 did not introduce artefactual heterogeneity at this section thickness. The sensitivity of this approach for the detection of minor subpopulations was calculated to be 13-16%, 17-18% and 10-11% for mono-, tri- and tetrasomic populations, respectively. Karyotypic heterogeneity was detected in two of the five tumours and, in one case where the populations where clustered morphologically, a minor population representing 18% was identified. Conclusions—Interphase cytogenetic analysis of sections from paraffin wax embedded material can be used for the detection of minor subpopulations in tumours. This approach will be of particular value in the assessment of the relation between human papillomavirus infection and tumour karyotype and in the analysis of intraepithelial neoplasia. Images PMID:16696090

The gene encoding the specific glycosyltransferases which catalyze the conversion of the H antigen to A or B antigens shows a slight but distinct variation in its allelic nucleotide sequence and can be divided into 6 genotypes when digested with specific restriction enzymes. We extracted DNA from formalin-fixed, paraffin-embedded tissues using SDS/proteinase K treatment followed by phenol/chloroform extraction. The sequence of nucleotides for the A, B and O genes was amplified by the polymerase chain reaction (PCR). DNA fragments of 128 bp and 200 bp could be amplified in the second round of PCR, using an aliquot of the first round PCR product as template. Degraded DNA from paraffin blocks stored for up to 10.7 years could be successfully typed. The ABO genotype was deduced from the digestion patterns with an appropriate combination of restriction enzymes and was compatible with the phenotype obtained from the blood sample. PMID:7947334

Background The use of DNA from archival formalin and paraffinembedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue. Methodology/Principal Findings Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample. Conclusions/Significance We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples. PMID:25133528

DNA from archival, formaldehyde fixed, paraffin wax embedded human tissue, suitable for amplification by the polymerase chain reaction (PCR), was obtained using a microwave method based on the capture of DNA by magnetic beads. Fragments of the α-1-antitrypsin gene (AAT) and the apolipoprotein E gene (APOE) were amplified successfully from human liver and brain tissue, respectively. This procedure provides a more rapid, simple and efficient method for reproducibly obtaining DNA from preserved tissue that has been kept in storage for up to 30 years. Images PMID:16696069

Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples. PMID:26981433

Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples. PMID:26981433

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffinembedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures (1). To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion (2). Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions

Background: Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities. Aims: To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples. Methods: DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes. Results: Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively. Aspergillus fumigatus DNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in another two cases. Conclusions: The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment. PMID:16254108

Infections by Nocardia spp. are generally regarded as opportunistic diseases in immunocompromised patients, but can also affect immunocompetent subjects. Such infections represent an important diagnostic challenge for clinicians and microbiologists, and diagnosis is frequently delayed or even conducted post mortem. A 54-year-old man was admitted to our hospital because of ventriculitis and relapsing brain abscess. Five months prior, this patient had undergone external ventricular drain and surgery for a cerebellar abscess. Histopathology demonstrated pyogenic inflammatory reaction, microbiologic investigations proved negative and empiric antimicrobial therapy was administered for a total of eight weeks. Six weeks later, the patient developed relapsing neurologic manifestations. On reviewing the patient's clinical history it emerged that the patient had suffered pneumonia two months prior to neurosurgery, treated with amoxicillin/clavulanate 3g a day and levofloxacin 500mg a day for three weeks. On the CNS relapsing manifestations, nocardiosis was suspected and DNA sequencing from the formalin-fixed paraffin-embedded cerebellar tissue collected during neurosurgery allowed diagnosis of Nocardia paucivorans infection. The patient received medical therapy for 11 months. At follow-up, eight months after treatment was discontinued, the patient was aymptomatic. Nocardia spp. infections need to be suspected not only in immunocompromised, but also in immunocompetent patients. Proper samples need to be collected for proper microbiologic investigations. Paraffin-embedded tissue genomic sequencing can be a useful tool for diagnosis of nocardiosis. PMID:27367327

We present a technique which allows the detection and chromosomal localization of DNA sequence copy number changes in solid tumor genomes from frozen sections and paraffinembedded, formalin fixed specimens. Based on comparative genomic hybridization and on universal DNA amplification procedures this technique is possible even if only a few tumor cells are available. We demonstrate the feasibility of this method to visualize complete and partial chromosome gains and losses and gene amplifications in archived solid tumor samples. PMID:8281155

Fluorescence in situ hybridization (FISH) has recently become important for pathological diagnosis. However, its practical applications is not widespread because FISH protocol with FFPE specimens is complicated. We report a dual detection method by overlapping FISH with fluorescent immunostaining on FFPE sections. This method is characterized by changing buffers for heat treatment without proteolytic enzyme treatment. Subsequent proteolytic enzyme treatment can be omitted using an antigen activation solution, pH9 (Nichirei Corporation), for heat treatment. After the pretreatment, dual detection was achieved by DNA FISH following RNA FISH and fluorescent immunostaining. This protocol visualized gene abnormalities and protein overexpression on the same sections. Of note, in poorly differentiated tumors containing both normal and tumor cells, the tumor cells were clearly identified on the sections, and FISH signals could be counted in these cells. In addition, HER2 mRNA overexpression and gene amplification were simultaneously detected in HER2-positive gastric cancer. Thus, this method should be widely applicable in clinical settings. PMID:26548243

Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology. To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes. In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the

In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis. PMID:20637756

In bone marrow transplantation (BMT) the detection of residual host lymphoid or haematopoietic cells surviving conditioning therapy is because of its association to graft-versus-host disease, graft-versus-leukemia reaction, and relapse of leukemia a matter of great interest. We studied the occurrence of this mixed lymphoid chimerism (MC) in the formol-fixed lymphatic tissue of lymph nodes and spleen from 21 autopsies after allogeneic sex-mismatched BMT (5 females, 16 males, survival 5 to 1140 days after BMT). In situ hybridisation with biotinylated centromer-specific anti-X- and anti-Y-chromosome probes was performed on pepsin-digested paraffinsections. The number of double X-, single X-, and Y-chromosome bearing cells was analysed microscopically. Because of artefacts only 14 cases remained for valid investigation. MC was detected in 6 cases (5 out of 11 males 5 days to 840 days and 1 out of 3 females 76 days after BMT). MC occurred after whole body irradiation with 10 Gy (n = 5) and 7 Gy (n = 1). In 1 autopsy relapse of leukemia caused host cell infiltration. Cases with MC did not express histological signs of acute or chronic graft-versus-host disease, but 5 out of 8 with complete lymphoid chimerism did. The sensitivity of interphase cytogenetics on paraffinembedded tissue is low. PMID:7534002

A modified fixative of formalin dichromate was combined with a cold embedding procedure for the preservation of bovine leucocyte surface antigens. Fourteen monoclonal antibodies recognizing seven bovine leucocyte surface antigens (BoCD1w2, BoCD4, BoCD8, BoWC1, BoWC3, BoWC4 and BoIgM) were applied as primary antisera in a sensitive avidin-biotin-peroxidase complex detection method. The staining results were compared with those obtained in cryostat and routinely formalin-fixed sections of corresponding tissue samples. Using the modified formalin dichromate fixative and the cold embedding procedure, all the leucocyte surface antigens tested were detectable immunohistologically in paraffinsections with a generally more distinct staining than in traditionally processed tissues. Morphological structures were better preserved than in cryostat sections but, to some extent, were poorer when compared with routinely formalinfixed tissues. However, this method suggests that there are only mild masking effects and provides an alternative to the use of unfixed material, particularly for morphological-immunohistochemical investigations. PMID:9248856

Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for proteomic studies. Multiple FFPE core biopsies can be assembled in a single block to form tissue microarrays (TMAs). We describe a protocol for analyzing protein in FFPE -TMAs using matrix-assisted laser desorption/ionization (MAL DI) imaging mass spectrometry (IMS). The workflow incorporates an antigen retrieval step following deparaffinization, in situ trypsin digestion, matrix application and then mass spectrometry signal acquisition. The direct analysis of FFPE -TMA tissue using IMS allows direct analysis of multiple tissue samples in a single experiment without extraction and purification of proteins. The advantages of high speed and throughput, easy sample handling and excellent reproducibility make this technology a favorable approach for the proteomic analysis of clinical research cohorts with large sample numbers. For example, TMA analysis of 300 FFPE cores would typically require 6 h of total time through data acquisition, not including data analysis. PMID:22011652

Terahertz (THz) spectroscopy constitute promising technique for biomedical applications as a complementary and powerful tool for diseases screening specially for early cancer diagnostic. The THz radiation is not harmful to biological tissues. As increased blood supply in cancer-affected tissues and consequent local increase in tissue water content makes THz technology a potentially attractive. In the present work, samples of healthy and adenocarcinoma-affected gastric tissue were analyzed using transmission time-domain THz spectroscopy (THz-TDS). The work shows the capability of the technique to distinguish between normal and cancerous regions in dried and paraffin-embedded samples. Plots of absorption coefficient α and refractive index n of normal and cancer affected tissues, are presented and the conditions for discrimination between normal and affected tissues are discussed.

In this work, samples of non-neoplastic and adenocarcinoma-affected human colon tissue samples were analyzed using multipoint transmission time-domain THz spectroscopy (THz-TDS) to sort out the contrast-contributing factors other than water, the main contrast mechanism factor in in-vivo or in freshly excised bio-tissue. Solving the electromagnetic inverse problem through THz-TDS and, analyzing the transmittance spectra that yielded the frequency-dependent absorption coefficient α and refractive index n of non-neoplastic and neoplastic tissues, we show that it is possible to distinguish between non-neoplastic and neoplastic regions in paraffin-embedded dehydrated. Results and discussion are presented.

The scattering properties and refractive indices (RI) of tissue are important parameters in tissue optics. These parameters can be determined from quantitative phase images of thin slices of tissue blocks. However, the changes in RI and structure of cells due to fixation and paraffinembedding might result in inaccuracies in the estimation of the scattering properties of tissue. In this study, three-dimensional RI distributions of cells were measured using digital holographic microtomography to obtain total scattering cross sections (TSCS) of the cells based on the first-order Born approximation. We investigated the slight loss of dry mass and drastic shrinkage of cells due to paraformaldehyde fixation and paraffinembedding removal processes. We propose a method to compensate for the correlated changes in volume and RI of cells. The results demonstrate that the TSCS of live cells can be estimated using restored cells. The percentage deviation of the TSCS between restored cells and live cells was only -8%. Spatially resolved RI and scattering coefficients of unprocessed oral epithelium ranged from 1.35 to 1.39 and from 100 to 450 cm-1, respectively, estimated from paraffin-embedded oral epithelial tissue after restoration of RI and volume.

Type and quality of sample preparation have significant impact on imaging mass spectrometry results. Though imaging of fresh-frozen tissues is considered to give the best results, they are incompatible with clinical practice, since routine diagnostics is most frequently performed using formalin-fixed tissues, and formalin-fixed paraffin-embedded material is a gold standard in histopathology. We aimed to assess utility of formalin-fixed tissue specimen processed without paraffinembedding (i.e., deep-frozen and cryo-sectioned) for MALDI imaging of both peptides and lipids. Peptide and lipid imaging was performed in fresh-frozen, FFPE and formalin-fixed/frozen samples of a mouse kidney, then composition of the resulting spectra was compared. We demonstrated similarity of spectra registered during peptide imaging in FFPE and formalin-fixed/frozen tissues, and similarity of spectra registered during lipid imaging in fresh-frozen and formalin-fixed/frozen material. Furthermore, molecular images of formalin-fixed/frozen tissue resembled the features of both fresh-frozen and FFPE tissue in the case of peptide imaging, and the features of fresh-frozen tissue in the case of lipid imaging. We conclude that tissue preserved by formalin fixation and processed without paraffinembedding can be considered as an alternative to both fresh-frozen and FFPE material. PMID:27001204

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry

Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffinembedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffinembedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffinembedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffinembedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives. PMID:26150155

Analysis of previously unknown genetic aberrations in solid tumors has become possible through the use of comparative genomic hybridization (CGH), which is based on competitive binding of tumor and control DNA to normal metaphase chromosomes. CGH allows detection of DNA sequence copy number changes (deletions, gains, and amplifications) on a genome-wide scale in a single hybridization. We describe here an improved CGH technique, which enables reliable detection of copy number changes in archival formalin-fixed paraffin-embedded tumor samples. The technique includes a modified DNA extraction protocol, which produces high molecular weight DNA which is necessary for high quality CGH. The DNA extraction includes a 3-day digestion with proteinase K, which remarkably improves the yield of high molecular weight DNA. Labeling of the test DNA with a directly fluorescein-conjugated nucleotide (instead of biotin labeling) improved significantly the quality of hybridization. Using the paraffin-block technique, we could analyze 70 to 90% of paraffin blocks, including very old samples as well as samples taken at autopsy. CGH from paraffin blocks was highly concordant (95%) with analyses done from matched freshly frozen tumor samples (n = 5 sample pairs; kappa coefficient = 0.83). The method described here has wide applicability in tumor pathology, allowing large retrospective prognostic studies of genetic aberrations as well as studies on genetic pathogenesis of solid tumors, inasmuch as premalignant lesions and primary and metastatic tumors can be analyzed by using archival paraffin-embedded samples. Images Figure 1 Figure 3 PMID:7992835

Cytogenetic studies in lymphomas classically require fresh or frozen tissue, whereas in many instances only paraffin-embedded biopsies are available. We applied an interphase FISH assay on nuclei extracted from thick paraffinsections to determine accuracy of molecular cytogenetics in such samples. Twenty-three lymphoma samples and 4 reactive lymph nodes were tested with various commercially available DNA probes, and hybridization patterns were compared with those obtained on frozen nuclei counterparts. Successful hybridization with all probes tested was observed for 23/27 (85%) paraffin-embedded tissues and for all (100%) frozen samples, and cut-off levels defining positivity were superimposable for both situations. Chromosome changes were detected in the same way, without any false-positive or false-negative cases. Hybridization signals observed on dewaxed samples were either those classically expected to define the relevant chromosome change or were atypical: all atypical changes could be demonstrated also into nuclei from the frozen counterpart. Moreover, all typical and atypical chromosome changes observed on frozen nuclei were also detected in paraffin-embedded tissues. Our study shows that our interphase FISH assay performed on paraffin-embedded samples is a valuable alternate to conventional methods to ascertain diagnosis of lymphomas as to include patients into therapeutic trials. PMID:15167011

Estrogen receptor (ER) analysis in breast cancer has been used in three clinical situations: to select patients with advanced breast cancer for hormonal therapy, as a prognostic parameter, and for selection of women with early breast cancer to adjuvant hormonal treatment. ER has traditionally been measured using labelled hormone in binding assays--often in dextran-coated charcoal assays (DCC). Monoclonal antibodies to ER has permitted development of a solid phase enzyme immunoassay (ER-EIA) used for quantitative determination of ER in tissue homogenates, and have also been used for determination of ER using an immunohistochemical assay in frozen sections (ER-ICA) or in formalin-fixed, paraffin-embedded tissue (ER-PAR). A large number of studies has compared ER-EIA with ER-DCC assays. There is a good linear correlation between the two types of assay but ER-EIA measure more ER and classify a larger fraction of tumors ER-positive than conventional ER assays. Lack of clinical data makes the significance of this uncertain. Numerous studies have reported on the correlation between ER-ICA and ER-DCC or ER-EIA. There is a good correlation among the assays on classification of ER status with a median 86% concordance, but a somewhat poorer correlation between semiquantified ER of immunohistochemical assays and ER determined by the quantitative methods (median coefficient of correlation 0.67). There is a large variation in the cut-off level for definition of ER-positive in immunohistochemical assays emphasizing the need for quality control studies. The major problem involved in ER analysis in paraffin-embedded tissue is a considerable loss of immunoreactivity compared to sections from frozen tissue. This can partly be overcome by modifications of the immunohistochemical technique using enzyme pretreatment and other amplification systems, but the sensitivity of ER-PAR remains lower than ER-ICA despite these modifications, and the ER status is less reliably determined in tumors

As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin fixed, paraffinembedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high quality DNA extraction from archival FFPE tissue specimen remains complex and time consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit; O.D 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with MSP can be used for epigenetic studies with the advantages of rapidity and high quality, and may contribute to the development of biomarkers in clinical studies. PMID:22449494

Dedifferentiated and differentiated liposarcoma are characterized by 12q15 chromosomal amplification. Comparative genomic hybridization is a powerful tool able to detect DNA copy number changes in the genome. This technique has been widely used in frozen tumors and in some studies in paraffin-embedded tumors fixed with formalin. The purpose of this study was to demonstrate the ability of CGH to detect DNA copy number changes in the genome when the DNA was extracted from tissues fixed with Holland Bouin's fluid. Sixteen liposarcoma tumors both frozen and fixed in Holland Bouin's fluid were characterized by CGH. Eighty-one percent of the main chromosomal alterations detected in the frozen liposarcomas (amp 12q15, amp 6q23, amp 1p32, amp 16q22, +7, +8) were detected in the corresponding fixed tumors. The limitation of this technique when using Holland Bouin's fluid extracted DNA compared with formalin-extracted DNA was the yield of analyzable samples. Eighty-one percent of tumors fixed with Holland Bouin's fluid (13/16) were analyzable compared with 100% of formalin-fixed tumors (4/4). This study demonstrates that comparative genomic hybridization is a useful tool even if only fixed tissues (formalin and Holland Bouin's fluid tissues) are available, and that it allows more tumors to be analyzed in retrospective studies. PMID:12960699

BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

In most hospitals word-wide, histopathological cancer diagnosis is currently based on formalin-fixed and paraffin-embedded (FFPE) tissues. In the last few years new approaches and developments in patient-tailored cancer therapy have raised the need to select more precisely those patients, who will respond to personalised treatments. The most efficient way for optimal therapy and patient selection is probably to provide a tumour-specific protein network portrait prior to treatment. The discovery and characterisation of deregulated signalling molecules (e.g. human epidermal growth factor receptor 2, mitogen-activated protein kinases) are very promising candidates for the identification of new suitable therapy targets and for the selection of those patients who will receive the greatest benefit from individualised treatments. The reverse phase protein array (RPPA) is a promising new technology that allows quick, precise and simultaneous analysis of many components of a network. Importantly it requires only limited amounts of routine clinical material (e.g. FFPE biopsies) and can be used for absolute protein measurements. We and other research groups have described successful protein extraction from routine FFPE tissues. In this manuscript we show how these recent developments might facilitate the implementation of RPPA in clinical trials and routine settings. PMID:19914823

Formalin-fixed paraffinembedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation. PMID:27583817

BRCA mutated ovarian cancers respond better to platinum-based therapy and to the recently approved PARP-inhibitors. There is the need for efficient and timely methods to detect both somatic and germline mutations using formalin-fixed paraffin-embedded (FFPE) tissues and commercially available technology. We used a commercial kit exploring all exons and 50bp exon-intron junctions of BRCA1 and BRCA2 genes, and semiconductor next-generation sequencing (NGS) on DNA from 47 FFPE samples of high-grade serous ovarian cancers. Pathogenic mutations were found in 13/47 (28%) cancers: eight in BRCA1 and five in BRCA2. All BRCA1 and two BRCA2 mutations were germline; three BRCA2 mutations were somatic. All mutations were confirmed by Sanger sequencing. To evaluate the performance of the NGS panel, we assessed its capability to detect the 6,953 variants described for BRCA1 and BRCA2 in ClinVar and COSMIC databases using callability analysis. 6,059 (87.1%) variants were identified automatically by the software; 829 (12.0%) required visual verification. The remaining 65 (0.9%) variants were uncallable, and would require 15 Sanger reactions to be resolved. Thus, the sensitivity of the NGS-panel was 99.1%. In conclusion, NGS performed with a commercial kit is highly efficient for detection of germline and somatic mutations in BRCA genes using routine FFPE tissue. PMID:26745875

As new technologies come within reach for the average cytogenetic laboratory, the study of chromosome structure has become increasingly more sophisticated. Resolution has improved from karyotyping (in which whole chromosomes are discernible) to fluorescence in situ hybridization and comparative genomic hybridization (CGH, with which specific megabase regions are visualized), array-based CGH (aCGH, examining hundreds of base pairs), and next-generation sequencing (providing single base pair resolution). Whole genome next-generation sequencing remains a cost-prohibitive method for many investigators. Meanwhile, the cost of aCGH has been reduced during recent years, even as resolution has increased and protocols have simplified. However, aCGH presents its own set of unique challenges. DNA of sufficient quantity and quality to hybridize to arrays and provide meaningful results is required. This is especially difficult for DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Here, we compare three different methods for acquiring DNA of sufficient length, purity, and "amplifiability" for aCGH and other downstream applications. Phenol-chloroform extraction and column-based commercial kits were compared with adaptive focused acoustics (AFA). Of the three extraction methods, AFA samples showed increased amplicon length and decreased polymerase chain reaction (PCR) failure rate. These findings support AFA as an improvement over previous DNA extraction methods for FFPE tissues. PMID:26126956

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens stored in pathology departments worldwide are an invaluable source for diagnostic purposes when fresh clinical material is unavailable as well as for retrospective molecular and epidemiological studies, especially when dealing with rare clinical conditions for which prospective collection is not feasible. Accurate detection of HPV infection in these specimens is particularly challenging because nucleic acids are often degraded and therefore, not suitable for amplification of larger fragments of the viral genome or viral gene transcripts. This review provides a brief summary of molecular methods for detecting alpha-HPV DNA/RNA in FFPE tissue specimens. We specifically address the key procedural and environmental factors that have the greatest impact on the quality of nucleic acids extracted from FFPE tissue specimens, and describe some solutions that can be used to increase their integrity and/or amplifiability. Moreover, commonly used methods for HPV DNA/RNA detection in FFPE tissue specimens are presented and discussed, focusing on studies using polymerase chain reaction as an HPV detection method and published after 1999. Finally, we briefly summarize our 22 years of experience with HPV detection in FFPE tissue specimens. PMID:26514313

Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving samples in RNAlater, and by formalin-fixation, paraffin-embedding on human soft tissue, using directly frozen samples as a control ("Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples" [1]). We here report the data from the analysis. The comparative analysis was performed on 24 colon mucosa biopsies, extracted from the sigmoideum of two gastroenterologically healthy participants for the purpose of this study. A set of biopsies were additionally stored for 30 min at room temperature prior to formalin-fixation. The samples were analyzed by high throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029. PMID:26937473

Newcastle disease (ND) is highly contagious and causes severe economic losses to the poultry industry due to high morbidity and mortality. In this report, we describe the detection of Newcastle disease virus (NDV) in formalin-fixed tissues from an outbreak of ND on broiler farms in Egypt. The affected birds experienced respiratory and/or nervous signs and a 75% mortality rate. Tissue samples were collected and placed in 10% neutral buffered formalin followed by embedding in paraffin. RNA was extracted from 80-microm formalin-fixed paraffin-embedded tissue blocks and recovered in 60 microl of elution buffer. All samples were negative for influenza virus by real-time reverse-transcription (RT)-PCR but positive for NDV. These flocks were known to have been vaccinated with a live NDV vaccine (LaSota strain). The nucleic acid sequences of the virus detected in this study were similar to those of a velogenic virus at its cleavage site 111GRRQKR*F117 and clustered with class II genogroup VII lineage of NDV, with a nucleotide sequence identity of 94%-99%. Although extraction and amplification of NDV from paraffin-embedded tissues from experimentally infected birds has been reported previously, this study reports on the use of RT-PCR on formalin-fixed tissues from actual field samples. PMID:24758123

Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving samples in RNAlater, and by formalin-fixation, paraffin-embedding on human soft tissue, using directly frozen samples as a control (“Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples” [1]). We here report the data from the analysis. The comparative analysis was performed on 24 colon mucosa biopsies, extracted from the sigmoideum of two gastroenterologically healthy participants for the purpose of this study. A set of biopsies were additionally stored for 30 min at room temperature prior to formalin-fixation. The samples were analyzed by high throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029. PMID:26937473

Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffinembedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material. PMID:26348873

Formalin-fixed paraffin-embedded (FFPE) tissue specimens constitute a highly valuable source of clinical material for retrospective molecular studies. However, metabolomic assessment of such archival material remains still in its infancy. Hence, there is an urgent need for efficient methods enabling extraction and profiling of metabolites present in FFPE tissue specimens. Here we demonstrate the methodology for isolation of primary metabolites from archival tissues; either fresh-frozen, formalin-fixed or formalin-fixed and paraffin-embedded specimens of mouse kidney were analysed and compared in this work. We used gas chromatography followed by mass spectrometry (GC/MS approach) to identify about 80 metabolites (including amino acids, saccharides, carboxylic acids, fatty acids) present in such archive material. Importantly, about 75% of identified compounds were detected in all three types of specimens. Moreover, we observed that fixation with formalin itself (and their duration) did not affect markedly the presence of particular metabolites in tissue-extracted material, yet fixation for 24h could be recommended as a practical standard. Paraffinembedding influenced efficiency of extraction, which resulted in reduced quantities of several compounds. Nevertheless, we proved applicability of FFPE specimens for non-targeted GS/MS-based profiling of tissue metabolome, which is of great importance for feasibility of metabolomics studies using retrospective clinical material. PMID:26348873

Summary Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA from pathology blocks by PCR and sequencing is an alternative approach to determine the etiology of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology, probably due to DNA damage by the tissue fixation. We used realtime PCR to quantify human and fungal DNA from Formalin-fixed, paraffinembedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing incubation temperature from 65°C to 90°C. An additional increase was documented by incubation for up to 6 hours at 90°C. The augmented amplification of fungal DNA was associated with improved species identification by sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy. PMID:22414380

Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrPCWD) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrPCWD burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrPRES in prion, and potentially other, protein misfolding disease states. PMID:27157060

Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected ∼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice. PMID:27414759

Traditional diagnostic detection of chronic wasting disease (CWD) relies on immunodetection of misfolded CWD prion protein (PrP(CWD)) by western blotting, ELISA, or immunohistochemistry (IHC). These techniques require separate sample collections (frozen and fixed) which may result in discrepancies due to variation in prion tissue distribution and assay sensitivities that limit detection especially in early and subclinical infections. Here, we harness the power of real-time quaking induced conversion (RT-QuIC) to amplify, detect, and quantify prion amyloid seeding activity in fixed paraffin-embedded (FPE) tissue sections. We show that FPE RT-QuIC has greater detection sensitivity than IHC in tissues with low PrP(CWD) burdens, including those that are IHC-negative. We also employ amyloid formation kinetics to yield a semi-quantitative estimate of prion concentration in a given FPE tissue. We report that FPE RT-QuIC has the ability to enhance diagnostic and investigative detection of disease-associated PrP(RES) in prion, and potentially other, protein misfolding disease states. PMID:27157060

The vast majority of human tissue specimens are formalin-fixed, paraffinembedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer’s disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer’s disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue. PMID:26487484

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS. PMID:26222067

To date the diagnosis of abdominal angiostrongyliasis (AA) depends on the histological identification of Angiostrongylus costaricensis (AC) in surgical specimens. However, microscopic evaluation is time consuming and often fails in identifying the parasite. We tested whether PCR might help in the diagnosis of AA by identifying parasite DNA in formalin-fixed paraffin-embedded (FFPE) tissue. We used primers based on DNA from Angiostrongilus cantonensis. Four groups of FFPE intestinal tissue were tested: (1) confirmed cases (n = 20), in which AC structures were present in the target tissue; (2) presumptive cases (n = 20), containing changes secondary to AC infection in the absence of AC structures; (3) negative controls (n = 3), consisting of normal colonic tissue; and (4) tissue affected by other parasitoses (n = 7), including strongyloidiasis, ascaridiasis, schistosomiasis, and enterobiasis. Most lesions of confirmed cases were located in small and/or large bowel (90%), as compared with presumptive cases, in which 70% of lesions were in appendix (P = 0.0002). When confronted with cases of other parasitoses, PCR showed sensitivity of 55%, specificity of 100% and positive predictive value of 100%. In presumptive cases PCR was positive in 4 (20%). All specimens from negative controls and other parasitoses were negative. In conclusion, the PCR technique showed intermediate sensitivity and optimal specificity, being clinically relevant when positive for abdominal angiostrongyliasis. It allowed a 20% gain in diagnosis of presumptive cases. PCR might help in the diagnosis of abdominal angiostrongyliasis, particularly when the pathologists are not experienced with such disease. PMID:24705328

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS. PMID:26222067

The background for this study was reports in the literature of stronger fluorescence observed visually for melanomas compared with benign naevi in formalin-fixed paraffin-embeddedsections. Our objective was to carry out a quantitative study of the phenomenon and to investigate if such an approach could be used in the detection of melanomas. Microscopic digital imaging was used to measure quantitatively the fluorescence intensity in specimens from 50 malignant melanomas, four basal cell carcinomas and 58 benign lesions. The mean fluorescence intensity of the melanomas was considerably higher than of the other lesions. For melanomas, the intensity depended both on the distance from the skin surface and the distance from the centre of the lesion. A simple algorithm based on the intensity threshold correctly classified the melanomas with a sensitivity of 74% and a specificity of 59%. Quantitative measurements of the fluorescence of the pigmented skin lesions fixed with formalin and embedded in paraffin can be a useful auxiliary tool for differentiating melanoma from other pigmented lesions histopathologically. PMID:11725203

The scattering properties and refractive indices (RI) of tissue are important parameters in tissue optics. These parameters can be determined from quantitative phase images of thin slices of tissue blocks. However, the changes in RI and structure of cells due to fixation and paraffinembedding might result in inaccuracies in the estimation of the scattering properties of tissue. In this study, three-dimensional RI distributions of cells were measured using digital holographic microtomography to obtain total scattering cross sections (TSCS) of the cells based on the first-order Born approximation. We investigated the slight loss of dry mass and drastic shrinkage of cells due to paraformaldehyde fixation and paraffinembedding removal processes. We propose a method to compensate for the correlated changes in volume and RI of cells. The results demonstrate that the TSCS of live cells can be estimated using restored cells. The percentage deviation of the TSCS between restored cells and live cells was only −8%. Spatially resolved RI and scattering coefficients of unprocessed oral epithelium ranged from 1.35 to 1.39 and from 100 to 450 cm−1, respectively, estimated from paraffinembedded oral epithelial tissue after restoration of RI and volume. PMID:25069007

The objective of the study was to develop an immunohistochemical (IHC) assay for rapid detection of Erysipelothrix rhusiopathiae. Serotypes 1a, 1b, and 2 are most frequently associated with clinical disease in pigs. Antiserum against serotypes 1a, 1b, and 2 was produced in rabbits, pooled, and applied to formalin-fixed, paraffin-embedded tissue sections of pigs (lungs, heart, spleen, and skin). The results obtained with the IHC assay were compared with direct culture on tissue samples from experimentally inoculated pigs either treated (n = 6) with antibiotics or untreated (n = 8) as well as on samples from field cases (n = 170) submitted to the Veterinary Diagnostic Laboratory at Iowa State University. The agreement between direct culture and IHC staining was found to be substantial. The results of the present study indicate that the IHC assay is highly sensitive and specific in detecting E. rhusiopathiae antigen in formalin-fixed, paraffin-embedded tissues. Results indicated that the IHC is particularly useful in cases in which pigs had been treated with antibiotics prior to submission and in which direct cultures of organs were negative. In addition, the IHC was found to be useful for detection of E. rhusiopathiae antigen in skin lesions, which are often culture negative. PMID:20093690

Purpose The removal of excessive melanin pigments that obscure ocular tissue morphology is important to address scientific questions and for differential diagnosis of ocular tumours based on histology. Thus, the goal of the present study was to establish an effective and fast melanin bleaching method for paraffin and frozen mouse and human ocular tissues. Methods Paraffin-embedded and frozen ocular specimens from mice and human donors were subjected to bleaching employing two methods. The first employed potassium permanganate (KMnO4) with oxalic acid, and the second 10% hydrogen peroxide (H2O2). To determine optimal bleaching conditions, depigmentation was carried out at various incubation times. The effect of diluents used for 10% H2O2 was assessed using phosphate-buffered saline (PBS), and deionized water. Three different slide types and two fixatives, which were ice-cold acetone with 80% methanol, and 4% paraformaldehyde (PFA) were used to determine the optimal conditions for better tissue adherence during bleaching. All tissues were stained in hematoxylin and eosin for histological evaluation. Results Optimal bleaching was achieved using warm 10% H2O2 diluted in PBS at 65°C for 120 minutes. Chromium-gelatin-coated slides prevented tissue detachment. Adherence of cryosections was also improved with post-fixation using 4% PFA and overnight air-drying at RT after cryosectioning. Tissue morphology was preserved under these conditions. Conversely, tissues bleached in KMnO4/oxalic acid demonstrated poor depigmentation with extensive tissue damage. Conclusions Warm dilute H2O2 at 65°C for 120 minutes rapidly and effectively bleached both cryo- and paraffinsections of murine and human ocular tissues. PMID:25025426

Archival samples represent a vast resource for identification of chemical and pharmaceutical targets. Previous use of formalin-fixed paraffin-embedded (FFPE) samples has been limited due to changes in RNA introduced by fixation and embedding procedures. Recent advances in RNA-seq...

Performance of immunofluorescence staining on archival formalin-fixed paraffin-embedded human tissues is generally not considered to be feasible, primarily due to problems with tissue quality and autofluorescence. We report the development and application of procedures that allowed for the study of a unique archive of thymus tissues derived from autopsies of individuals exposed to atomic bomb radiation in Hiroshima, Japan in 1945. Multiple independent treatments were used to minimize autofluorescence and maximize fluorescent antibody signals. Treatments with NH3/EtOH and Sudan Black B were particularly useful in decreasing autofluorescent moieties present in the tissue. Deconvolution microscopy was used to further enhance the signal-to-noise ratios. Together, these techniques provide high-quality single- and dual-color fluorescent images with low background and high contrast from paraffin blocks of thymus tissue that were prepared up to 60 years ago. The resulting high-quality images allow the application of a variety of image analyses to thymus tissues that previously were not accessible. Whereas the procedures presented remain to be tested for other tissue types and archival conditions, the approach described may facilitate greater utilization of older paraffin block archives for modern immunofluorescence studies. PMID:26392518

We present a strategy for the evaluation of numerical copy number changes of DNA segments within a solid tumor genome that allows the correlation of microscopic phenotype with genotype in formalin-fixed, paraffin-embedded tumor material. Cells from a human testicular germ cell tumor and adjacent tissue areas with normal seminiferous tubules were selected separately from microscopically analyzed histological tissue sections, and DNA was extracted from the selected areas. After universal DNA amplification, the amplification products were subjected to comparative genomic hybridization. The results confirmed balanced chromosome copy numbers for the normal tissue area, although the analysis of the tumor tissue area revealed numerous gains and losses of chromosome segments. The comparative genomic hybridization results were used to select DNA probes for interphase cytogenetics on serial sections. We conclude that this technique allows the screening of selected tissue areas for numerical DNA alterations, thus enabling a direct phenotype-genotype comparison. Images Figure 1 Figure 2 Figure 4 PMID:7778673

Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin-fixed, paraffin-embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumour banks. Once samples are fixed in formalin, the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study, we have evaluated the ability of the whole genome DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay from Illumina to perform transcriptomic analysis of archived breast tumour tissue in formalin-fixed, paraffin-embedded (FFPE) blocks. We profiled 76 familial breast tumours from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r(2) = 0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumour molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterization of many diseases. PMID:20593485

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology. PMID:26921540

Re-staining of formalin fixed paraffinsections sometimes is required and this requires prior de-staining. Some simple and effective protocols for de-staining are described. Mucihematoxylin and mucicarmine can be removed with acid alcohol. Zirconyl hematoxylin can be removed with periodic acid or Sinha's fixative. Alcian blue can be removed with 5% trifluoroacetic acid in dichloromethane. Colloidal iron can be bleached in 1% household bleach in alcohol. PAS can be removed with hydrogen peroxide or ammonium hydroxide. With few exceptions, de-stained sections can be re-stained with mucihematoxylin, PAS or Gabe's trichrome. PMID:20001228

Microscopic examination of a blood clot expelled by a physically and mentally disabled woman taken to the emergency room because of genital bleeding revealed the presence of chorionic villi encircled by decidua, hemorrhage, and necrosis. In order to identify the father of the product of conception, sections of formalin-fixed, paraffin-embedded abortion material were subjected to laser microdissection: DNA extraction from chorionic villi selectively isolated from the surrounding tissues allowed successful STR-typing of fetal cells, which was otherwise prevented by excess maternal DNA. The large number of homozygous genotypes in the fetal profile suggested incestuous paternity. Analysis of reference DNA samples from male relatives excluded the woman's father, paternal grandfather, and maternal grandfather, whereas the obligate paternal alleles of the fetus were constantly present in the genotypes of the woman's brother, clearly demonstrating brother-sister incest (probability of paternity > 99.99999%). PMID:16423229

An immunohistological study of paraffin wax embedded tissue from three cases of plasmacytoid monocyte neoplasms, using a panel of antibodies which react with fixation resistant leucocyte markers, is reported. This neoplasm was found to have a distinctive antigenic profile, being negative for CD3 and elastase, but positive for CD43 and CD68. This immunological phenotype, coupled with its characteristic morphological features, should facilitate the recognition of this rare neoplasm in routinely processed tissue. Furthermore, the term "plasmacytoid monocyte sarcoma" is proposed to designate it because it is inappropriate to refer to it as a lymphoma. As all cases have been associated with a myeloproliferative disorder (usually an acute or chronic myeloid leukaemia), these tumours probably represent the accumulation in lymphoid tissue of neoplastic cells which have differentiated along the plasmacytoid monocyte pathway. Images PMID:1890195

DNA extraction and PCR amplification from paraffin wax embedded bone tumour specimens present several difficulties, firstly, because of the abundant matrix they contain and, secondly, because decalcification often causes degradation of DNA. In this report, comparative studies were carried out to determine the most efficient method for DNA extraction and PCR amplification from such specimens. The results indicated that nested PCR produced appropriate strong reaction products with minimal background contamination. A method for DNA extraction from paraffin wax embedded bone tissue and a nested PCR-SSCP technique have been developed for use in such diagnostic specimens. PMID:16696068

Formalin-fixed paraffin-embedded (FFPE) tissues are a valuable source of DNA with which to perform large retrospective studies on the epidemiology of HPV infection. Five different DNA extraction protocols were carried out to evaluate the DNA obtained from FFPE samples with polymerase chain reaction (PCR) using two primer sets to amplify a constitutive human gene, β-globin, and two primer sets to detect the L1 and E6 HPV genes. From the five DNA extraction protocols evaluated, the best results were obtained with protocol A, corresponding to a crude extract from the sample. With the procedures described herein, we were able to amplify DNA extracted from archival paraffin blocks stored for six years. However, the amplification products were more efficiently obtained with primers that amplified shorter fragments. This result indicates that a major factor limiting the extraction process in these samples is DNA fragmentation, a factor that will naturally vary between the different specimens evaluated. Also, depending upon the extraction method, PCR amplification of a human gene does not necessarily guarantee the successful extraction of viral DNA. In conclusion, different DNA and HPV detection methods can significantly influence the results. Therefore, the DNA extraction methods and primers used for DNA amplification in fixed tissues need to be chosen carefully, depending on the specific requirements of the study being carried out. PMID:25444238

AIMS: To determine, by in situ immunohistochemistry, whether ovarian carcinomas have increased expression of DNA topoisomerase I. METHODS: Paraffin wax blocks obtained from 15 samples of normal human tissues and from 14 cases of ovarian cancer were cut on to glass slides and immunohistochemically stained for topoisomerase I. The primary antibody was a mouse monoclonal that recognises topoisomerase I in western blots. Colour was detected using a peroxidase system with diaminobenzidine as the chromogen. The expression of topoisomerase I in the tissues and tumours was graded subjectively from 0 to 3+ based on the colour intensity of the immunostain. RESULTS: In normal tissues, topoisomerase I expression was strongest in the mucosal lymphocytes in the gastrointestinal tract and in the germinal centres of the tonsil. Weak topoisomerase I staining was found in the columnar epithelium of the gastrointestinal tract and in squamous mucosa. In the series of ovarian carcinomas, raised topoisomerase I was observed in 43% (6 of 14) of the tumours. Of the tumours with raised topoisomerase I, only three contained a population of rapidly cycling cells. Therefore, 21% of our series of ovarian carcinomas (3 of 14) had raised topoisomerase I expression and were proliferating rapidly. CONCLUSIONS: Topoisomerase I expression in formalin fixed, paraffin wax embedded human tissues can be evaluated by immunohistochemical staining. Increases in topoisomerase I occur in some cases of ovarian cancer. Images PMID:9497914

A proper extraction method from formalin-fixed paraffin-embedded (FFPE) blocks is essential to obtain DNA of satisfactory quality/quantity. We compared the effectiveness of eight commercially available kits for DNA extraction based on 10 FFPE tissues. Kits differed significantly in terms of DNA yield, purity, and quality. Using the QIAamp DNA FFPE Tissue Kit (Qiagen) and the ReliaPrep FFPE gDNA Miniprep System (Promega), we obtained DNA of the highest quality and acceptable quantity. We also demonstrated that overnight digestion of samples usually improved DNA yield and/or purity. For precious or limited material, double elution is recommended for obtaining up to 42% higher amount of DNA. PMID:25640584

Coccidioides immitis and C. posadasii are the etiologic agents of coccidioidomycosis, an endemic fungal disease of the Americas. In Colombia, this mycosis is uncommon, and only five cases, two of them imported, have been documented.By means of DNA sequencing, C. immitis was identified in formalin-fixed, paraffin-embedded archival tissues samples from the 5th Colombian patient diagnosed in 1997. The patient was born in Pinto, Department of Magdalena, and had never visited other geographic regions, a reason to consider that the mycosis had been acquired locally.This species is primarily found in California although it has been occasionally reported in other geographic areas such as Mexico and Brazil. This is the first indigenous report of C. immitis-associated coccidioidomycosis in a Colombian patient. PMID:25908652

Environmental stresses can alter immunoreactivity of biomarkers in stored tissue sections. The effect of temperature and lighting on 49 cellular or microbial antigens was evaluated in 4 serial paraffinsections, cut 12 months, 10 months, 8 months, 5 months, 3 months, 1 month, 3 days, and 1 day before immunohistochemistry. Slides were stored at room temperature (RT) in the dark, at 4°C in the dark, at RT under fluorescent light, or at RT with windowpane exposure to sunlight. Immunohistochemistry was performed simultaneously in an automated immunostainer. Immunoreactivity was compared with that in the corresponding 1-day-old section and scored as 4 (<10% reduction), 3 (10%-25% reduction), 2 (26%-60% reduction), 1(>60% reduction), or 0 (no reactivity). Any loss of immunoreactivity was proportional to the tissue section age and was least in sections stored in the dark. Immunoreactivity was only completely lost in light-exposed sections and as early as 1 month for CD45. Other markers with complete loss of immunoreactivity were bovine viral diarrhea virus, CD18 (only with fluorescent light), CD31, CD68, canine parvovirus, chromogranins, and thyroid transcription factor-1. Markers with complete loss after light exposure also had reduced immunoreactivity when stored in the dark, as early as day 3. Eight markers (Bartonella spp, CD11d, high molecular weight cytokeratins, feline coronavirus, GATA-4, insulin, p63, progesterone receptor) had minimal decrease in immunoreactivity, regardless of treatment. In conclusion, light-induced antigen decay (tissue section aging) is antigen dependent and could explain unexpectedly weak or negative immunohistochemical reactions in stored paraffinsections. PMID:23435571

Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffinembedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended. PMID:24933424

The molecular mechanism responsible for Ewing's Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis. PMID:27144561

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections. PMID:20661639

In our previous report, we described a new fixation and paraffin-embedding method (the AMeX method) that preserves many of the antigens that are normally destroyed by routine formalin fixation. The current study was conducted to examine the preservation of high-molecular-weight DNA in tissues processed by this method. DNA was extracted from AMeX-processed tissue sections after deparaffinization by the same method as that used to extract DNA from fresh tissues. The total amounts of DNA extracted from 10 mg each in wet weight of AMeX-processed and fresh mouse liver tissues were identical. In tissues of malignant lymphoma, the total amount of spooled DNA extracted from 50 sections, each 20 microns thick, was about 8 micrograms/mm2. The electrophoretic pattern of DNA digested with restriction endonucleases on agarose gel from AMeX-processed tissue sections did not differ from that of fresh materials. Southern blot hybridization analysis also revealed that the mobility of specific DNA fragments was identical for AMeX-processed and fresh tissues. The AMeX method was thus proved to be a versatile multipurpose tissue-processing procedure, which is expected to provide important information regarding the correlation between morphology, phenotypic expression, and gene alteration. Images Figure 3 Figure 4 Figure 5A Figure 5B PMID:2407122

Following an intraperitoneal injection of tritiated thymidine to neonatal mice, livers and spleens were removed and their labelling indices were derived autoradiographically. This was done in a number of ways: (1) from tissue imprints on gelatinised glass slides; (2) from tissue embedded in JB4 plastic sectioned at thicknesses of 2, 5 and 7 micron; and (3) from tissue embedded in paraffin wax and sectioned at 7 micron. The results show that the indices from the JB4 embeddedsections increase as the section thickness decreases, and that this relationship persists down to the notional section thickness of zero in the tissue imprints (in which all the cells are in contact with the autoradiographic emulsion). Indices from the 7 micron paraffin wax embeddedsections are surprisingly close to the values from the imprints, are higher than indices from the 5 and 7 micron JB4 embeddedsections, and are not significantly different (at the 2% level) from those from 2 micron JB4 embeddedsections. Possible reasons for these results are discussed in respect of the autoradiographic process and in relationship to various mathematical correction factors which have been proposed to take account of beta-particle self-absorption in thick sections. It is concluded that none of these correction factors is of value and that the embedding medium has an important effect on the observed labelling indices. Comparisons between labelling indices, therefore, should be made only when they are derived from similarly embedded material at the same section thickness. PMID:3908424

Formalin-fixed paraffin-embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin-induced alternations on a proteome-wide level. We compared LC-MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2-6% of all peptide-spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar. PMID:25825003

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3–16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3–16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis. PMID:26580073

For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffinembedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is oftentimes degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. Using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 gene (FGFR2) that is amplified in a gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas. PMID:23682346

For the analysis of cancer, there is great interest in rapid and accurate detection of cancer genome amplifications containing oncogenes that are potential therapeutic targets. The vast majority of cancer tissue samples are formalin fixed and paraffinembedded (FFPE) which enables histopathological examination and long term archiving. However, FFPE cancer genomic DNA is oftentimes degraded and generally a poor substrate for many molecular biology assays. To overcome the issues of poor DNA quality from FFPE samples and detect oncogenic copy number amplifications with high accuracy and sensitivity, we developed a novel approach. Our assay requires nanogram amounts of genomic DNA, thus facilitating study of small amounts of clinical samples. Using droplet digital PCR (ddPCR), we can determine the relative copy number of specific genomic loci even in the presence of intermingled normal tissue. We used a control dilution series to determine the limits of detection for the ddPCR assay and report its improved sensitivity on minimal amounts of DNA compared to standard real-time PCR. To develop this approach, we designed an assay for the fibroblast growth factor receptor 2 gene (FGFR2) that is amplified in a gastric and breast cancers as well as others. We successfully utilized ddPCR to ascertain FGFR2 amplifications from FFPE-preserved gastrointestinal adenocarcinomas. PMID:23682346

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffinembedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R(2) = 0.94) and immuno-MRM (R(2) = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good quality and data of fresh frozen and FFPE tissues from healthy individuals were included as baseline controls in our data analysis. Our analysis showed a 91% overlap between CNV detection by microarray with FFPE tissues and chromosomal abnormality detection by karyotyping with fresh tissues on 8 cases of lymphoma samples. The CNV overlap between matched frozen and FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The analysis was further validated by Fluorescence In Situ Hybridization on these samples using probes specific for BRAF and CITED2. The results suggested that the modified method using Affymetrix Cytoscan HD chip gave rise to a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE microarray methodology may hold promise for broad application of CNV analysis on clinical samples. PMID:24699316

The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated. PMID:26403093

We determined the best extraction buffer for proteomic investigation using formalin-fixation and paraffin-embedded (FFPE) specimens. A Zwittergent 3-16 based buffer, sodium dodecyl sulfate (SDS)-containing buffer with/without polyethylene glycol 20000 (PEG20000), urea-containing buffer, and FFPE-FASP protein preparation kit were compared for protein extraction from different types of rat FFPE tissues, including the heart, brain, liver, lung, and kidney. All of the samples were divided into two groups of laser microdissected (LMD) and non-LMD specimens. For both kinds of specimens, Zwittergent was the most efficient buffer for identifying peptides and proteins, was broadly applicable to different tissues without impairing the enzymatic digestion, and was well compatible with mass spectrometry analysis. As a high molecular weight carrier substance, PEG20000 improved the identification of peptides and proteins; however, such an advantage is limited to tissues containing submicrograms to micrograms of protein. Considering its low lytic strength, urea-containing buffer would not be the first alternative for protein recovery. In conclusion, Zwittergent 3-16 is an effective buffer for extracting proteins from FFPE specimens for downstream proteomics analysis. PMID:26580073

Cystic echinococcosis (CE) caused by Echinococcus granulosus remains a serious problem worldwide for issues relating to public health and the economy. The most predominantly affected sites are the liver and the lungs, but other organs such as the heart, the spleen and the peritoneum can also be infected. Access to cysts from uncommon sites has limited genomic and molecular investigations. In the present study, genotypes of E. granulosus sensu lato were identified from formalin-fixed paraffin-embedded tissues (FF-PETs) implicated in human CE. Tissue samples were obtained from 57 patients with histologically confirmed CE. DNA samples were analysed using Egss 1 polymerase chain reaction (PCR) specific to the mitochondrial 12S rRNA gene of E. granulosus sensu stricto. All cysts were typed as E. granulosus sensu stricto with up to 35% of the liver and 16.6% of lungs being the most frequently infected, and up to 48.4% of samples being from rare sites. No correlation was found between cyst site and either the gender or the age of patients. This study demonstrates the possibility of exploiting atypical cysts using FF-PET samples and highlights the predominance of E. granulosus sensu stricto species in the Tunisian population, even in unusual infection sites. PMID:26190231

In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomolecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings. PMID:26354930

Biomarkers from tissue-based proteomic studies directly contribute to defining disease states as well as promise to improve early detection or provide for further targeted therapeutics. In the clinical setting, tissue samples are preserved as formalin-fixed paraffin-embedded (FFPE) tissue blocks for histological examination. However, proteomic analysis of FFPE tissue is complicated due to the high level of covalently cross-linked proteins arising from formalin fixation. To address these challenges, we developed well-based reverse-phase protein array (RPPA). This approach is a robust protein isolation methodology (29.44 ± 7.8 μg per 1 mm(3) of FFPE tissue) paired with a novel on electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots, demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, dried up and vacuum-sealed, remain stable up to 2 months at room temperature. This methodology is directly applicable to FFPE tissue and presents the direct opportunity of addressing hypothesis within clinical trials and well-annotated clinical tissue repositories. PMID:26043998

The aim of this retrospective study was to detect selected pathogens in pneumonic lung tissue of dogs of different age groups by immunohistochemistry (IHC), in situ hybridisation (ISH) or polymerase chain reaction (PCR) in order to get information about their involvement in pneumonia formation. In archived formalin-fixed and paraffin wax-embedded lung samples from 68 cases with the clinical and histologic diagnosis of pneumonia the histological pattern of pneumonia was re-evaluated and the samples were further investigated for the following infectious agents: canine distemper virus (CDV), canine adenovirus type 2 (CAV-2), canine respiratory coronavirus (CRCoV), Bordetella (B.) bronchiseptica, Pasteurella (P.) multocida, Mycoplasma spp., and Pneumocystis spp. In 47.1% of the samples at least one of the featured respiratory pathogens was detected. In 31.3% of these positive samples more than one pathogen could be found. The correct detection of CDV had been achieved in ten out of eleven positive cases (90.9%) upon initial investigation, but the presence of bacterial pathogens, like B. bronchiseptica (10 cases) and P. multocida (17 cases) had been missed in all but one case. While CDV and CRCoV infections were exclusively found in dogs younger than one year, the vast majority of infections with P. multocida and B. bronchiseptica were both common either in dogs younger than 4 months or older than one year. Thus, this retrospective approach yielded valuable data on the presence, absence and prevalence of certain respiratory pathogens in dogs with pneumonia. PMID:26919147

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease. PMID:16311361

Translating whole-exome sequencing (WES) for prospective clinical use may have an impact on the care of patients with cancer; however, multiple innovations are necessary for clinical implementation. These include rapid and robust WES of DNA derived from formalin-fixed, paraffin-embedded tumor tissue, analytical output similar to data from frozen samples and clinical interpretation of WES data for prospective use. Here, we describe a prospective clinical WES platform for archival formalin-fixed, paraffin-embedded tumor samples. The platform employs computational methods for effective clinical analysis and interpretation of WES data. When applied retrospectively to 511 exomes, the interpretative framework revealed a 'long tail' of somatic alterations in clinically important genes. Prospective application of this approach identified clinically relevant alterations in 15 out of 16 patients. In one patient, previously undetected findings guided clinical trial enrollment, leading to an objective clinical response. Overall, this methodology may inform the widespread implementation of precision cancer medicine. PMID:24836576

The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

In label-free Fourier-transform infrared histology, spectral images are individually recorded from tissue sections, pre-processed and clustered. Each single resulting color-coded image is annotated by a pathologist to obtain the best possible match with tissue structures revealed after Hematoxylin-Eosin staining. However, the main limitations of this approach are the empirical choice of the number of clusters in unsupervised classification, and the marked color heterogeneity between the clustered spectral images. Here, using normal murine and human colon tissues, we developed an automatic multi-image spectral histology to simultaneously analyze a set of spectral images (8 images mice samples and 72 images human ones). This procedure consisted of a joint Extended Multiplicative Signal Correction (EMSC) to numerically deparaffinize the tissue sections, followed by an automated joint K-Means (KM) clustering using the hierarchical double application of Pakhira-Bandyopadhyay-Maulik (PBM) validity index. Using this procedure, the main murine and human colon histological structures were correctly identified at both the intra- and the inter-individual levels, especially the crypts, secreted mucus, lamina propria and submucosa. Here, we show that batched multi-image spectral histology procedure is insensitive to the reference spectrum but highly sensitive to the paraffin model of joint EMSC. In conclusion, combining joint EMSC and joint KM clustering by double PBM application allows to achieve objective and automated batched multi-image spectral histology. PMID:26872124

Immunolabeling of tissue sections requires careful optimization of protocols in order to achieve accurate and consistent data. Multiple immunolabeling is desirable when determining the exact location and phenotype of cell populations in the same cellular compartment. 5-bromodeoxyuridine (BrdU)-immunolabeling is commonly used to assess cellular proliferation in vitro. However, the technical limitations of standard methods preclude multiple antigen immunolabeling. The aim was therefore to develop a robust protocol for simultaneous labeling using anti-BrdU and a melanocyte-specific marker in formalin-fixed paraffin-embedded (FFPE) skin samples. Human skin samples were obtained from patients undergoing elective plastic surgery. The tissue was incubated with BrdU, and a standard sample procedure for FFPE tissue was used. Heat-induced antigen retrieval was performed in a conventional pressure cooker, followed by immunolabeling with anti-BrdU and anti-Melan A/MART-1 antibodies. Fluorescent-conjugated secondary antibodies were used for signal detection. We have demonstrated both proliferating cells (BrdU-immunopositive) and melanocytes (Melan A/MART-1-immunopositive) in the basal compartment of the epidermis in our skin samples. Successful double labeling requires heat-induced epitope retrieval to replace the harsh pretreatment protocols of standard BrdU immunolabeling methods. We have optimized a robust protocol for the double labeling of proliferating cells and cells bearing melanocyte-specific antigens (melanocytes and/or melanoblasts) in FFPE human skin samples. PMID:22531682

Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases. PMID:8406434

Formalin-fixed paraffin-embedded (FFPE) tissue is a rich source of clinically relevant material that can yield important translational biomarker discovery using proteomic analysis. Protocols for analyzing FFPE tissue by LC-MS/MS exist, but standardization of procedures and critical analysis of data quality is limited. This study compared and characterized data obtained from FFPE tissue using two methods: a urea in-solution digestion method (UISD) versus a commercially available Qproteome FFPE Tissue Kit method (Qkit). Each method was performed independently three times on serial sections of homogenous FFPE tissue to minimize pre-analytical variations and analyzed with three technical replicates by LC-MS/MS. Data were evaluated for reproducibility and physiochemical distribution, which highlighted differences in the ability of each method to identify proteins of different molecular weights and isoelectric points. Each method replicate resulted in a significant number of new protein identifications, and both methods identified significantly more proteins using three technical replicates as compared to only two. UISD was cheaper, required less time, and introduced significant protein modifications as compared to the Qkit method, which provided more precise and higher protein yields. These data highlight significant variability among method replicates and type of method used, despite minimizing pre-analytical variability. Utilization of only one method or too few replicates (both method and technical) may limit the subset of proteomic information obtained. PMID:26306679

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues. PMID:25965788

Glioblastoma is the most common malignant brain tumor accounting for more than 54 % of all gliomas. Despite aggressive treatments, median survival remains less than 1 year. This might be due to the unavailability of effective molecular diagnostic markers and targeted therapy. Thus, it is essential to discover molecular mechanisms underlying disease by identifying dysregulated pathways involved in tumorigenesis. Notch signaling is one such pathway which plays an important role in determining cell fates. Since it is found to play a critical role in many cancers, we investigated the role of Notch genes in glioblastoma with an aim to identify biomarkers that can improve diagnosis. Using real-time PCR, we assessed the expression of Notch genes including receptors (Notch1, Notch2, Notch3, and Notch4), ligands (JAG1, JAG2, and DLL3), downstream targets (HES1 and HEY2), regulator Deltex1 (DTX1), inhibitor NUMB along with transcriptional co-activator MAML1, and a component of gamma-secretase complex APH1A in 15 formalin-fixed paraffin-embedded (FFPE) patient samples. Relative quantification was done by the 2(-ΔΔCt) method; the data are presented as fold change in gene expression normalized to an internal control gene and relative to the calibrator. The data revealed aberrant expression of Notch genes in glioblastoma compared to normal brain. More than 85 % of samples showed high Notch1 (P = 0.0397) gene expression and low HES1 (P = 0.011) and DTX1 (P = 0.0001) gene expression. Our results clearly show aberrant expression of Notch genes in glioblastoma which can be used as putative biomarkers together with histopathological observation to improve diagnosis, therapeutic strategies, and patient prognosis. PMID:26662803

Formalin fixing with paraffinembedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing. PMID:26641479

Formalin fixing with paraffinembedding (FFPE) has been a standard sample preparation method for decades, and archival FFPE samples are still very useful resources. Nonetheless, the use of FFPE samples in cancer genome analysis using next-generation sequencing, which is a powerful technique for the identification of genomic alterations at the nucleotide level, has been challenging due to poor DNA quality and artificial sequence alterations. In this study, we performed whole-exome sequencing of matched frozen samples and FFPE samples of tissues from 4 cancer patients and compared the next-generation sequencing data obtained from these samples. The major differences between data obtained from the 2 types of sample were the shorter insert size and artificial base alterations in the FFPE samples. A high proportion of short inserts in the FFPE samples resulted in overlapping paired reads, which could lead to overestimation of certain variants; >20% of the inserts in the FFPE samples were double sequenced. A large number of soft clipped reads was found in the sequencing data of the FFPE samples, and about 30% of total bases were soft clipped. The artificial base alterations, C>T and G>A, were observed in FFPE samples only, and the alteration rate ranged from 200 to 1,200 per 1M bases when sequencing errors were removed. Although high-confidence mutation calls in the FFPE samples were compatible to that in the frozen samples, caution should be exercised in terms of the artifacts, especially for low-confidence calls. Despite the clearly observed artifacts, archival FFPE samples can be a good resource for discovery or validation of biomarkers in cancer research based on whole-exome sequencing. PMID:26641479

Background: Cervical cancer is one of the important reasons of mortality among females. Prevention, early diagnosis and immediate treatment can affect the rate of mortality in this cancer and several epidemiological studies have shown a strong relationship between human papilloma viruses (HPVs) and cervical cancer. Objectives: The present study was conducted to survey HPV infections in a women population with cervical cancer and cervical dysplasia/metaplasia in southwest of Iran. Materials and Methods: 72 paraffin-embedded cervical biopsies which had been previously archived from women with cervical cancer and cervical dysplasia were examined by polymerase chain reaction (PCR). Afterward, the detected HPV strains were typed by restriction fragment length polymorphism (RFLP) analysis of PCR amplicons. Results: 60 out of 72 samples had necessary requirements and HPV DNA was detected in 43.3% of these samples. Most HPV positive samples belonged to women aged from 48 to 63 years. On the other hand, HPV infection among patients with squamous cell carcinoma (SCC) was 48.78% and in women with dysplasia/metaplasia was 26.66%. The most prevalent type of the human papilloma virus was HPV16 (100%). Conclusions: Knowing the most prevalent type of the human papilloma viruses circulating in the population (HPV16) can be applied in the future screening and managing programs of this major disease and also in vaccination against the prevalent types of the virus. Meanwhile, it seems that more studies should be performed to determine the role of different risk factors involved in development of the disease, especially those related with social behaviors and traditions with respect to different areas. PMID:27366309

The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected. PMID:24727804

We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits. PMID:25991403

Aim—To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. Methods—DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the ß globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. Results—Microwave extraction showed the highest positive rate for ß globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp ß globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 ß globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the ß globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. Conclusions—HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered. Key Words: cervical cancer • DNA extraction • polymerase chain reaction PMID:11328843

The NSABP B-32 trial is examining whether patients with initially negative sentinel lymph nodes (SLNs) who have occult metastases detected on deeper levels and cytokeratin immunohistochemistry (CK-IHC) stains are at risk for regional or distant metastases. The experimental B-32 protocol was designed to detect metastases larger than 1.0 mm by examining sections approximately 0.5 and 1.0 mm deeper into the paraffin blocks (2 levels; wide spacing). This pilot quality assurance study compares detection rates to a comprehensive protocol designed to detect metastases larger than 0.2 mm (multilevel; narrow spacing). All SLNs were sectioned grossly at close to 2.0 mm and all sectionsembedded in paraffin blocks. For clinical treatment, a single H&E section was examined from each block. For 54 cases with 1–5 SLNs and all SLNs negative, additional CK-IHC sections were evaluated every 0.18 mm through the block until no tissue remained. 20 of 176 (11.4%) blocks harbored occult metastases; the B-32 protocol detected metastases in 11 blocks (6.3%) and 9 additional blocks (5.1%) with metastases were detected on sections that would not have been evaluated (p=0.002; correlated proportions). Median number of levels examined per block on the comprehensive protocol was 11 (range 3–26); the B-32 protocol was fixed at 2 levels (median 2; range 1–2). Median thickness of node sections in the block was 2.1 mm (range 0.7–4.8 mm) and the modal thickness was 2.3 mm. Although more comprehensive sectioning of SLNs detects additional micrometastases, the data suggest diminishing returns and reduced cost effectiveness for the comprehensive strategy. PMID:19730364

Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9137080

The distribution of chemical elements in the normal human cornea was studied by energy dispersive x-ray analysis and scanning electron microscopy of routinely prepared paraffinsections. Calcium, phosphorus, and sulfur were consistently present in quantities above background and varied in concentration regionally. Analysis of fresh-frozen tissue, an approximation of the in vivo state, gave a similar elemental profile to paraffinsections, except for the loss of diffusable electrolytes in the latter. After fixation, S was the most abundant element and was highest in Descemet's membrane. Corneas with granular, lattice, macular, and Fuchs endothelial dystrophies, band keratopathy, and spheroidal degeneration were also examined. Characteristic patterns of abnormal S and Ca distribution were found in each of the dystrophies. The relative proportions of Ca, P, and S gave diagnostic profiles for distinguishing band keratopathy and spheroidal degeneration.

The diagnosis of T-cell lymphoproliferative disorders, which frequently involve the skin and other extranodal sites, is often problematic because of the difficulty in establishing clonality in paraffin-embedded tissue. To this end, we developed a simple, nonradioactive method to detect T-cell receptor γ (TCR-γ) gene rearrangements by polymerase chain reaction single-strand conformational polymorphism (PCR-SSCP) in paraffin-embedded tissue. Jurkat and HSB-2 cell lines and peripheral blood samples from normal individuals were used as monoclonal and polyclonal controls, respectively. DNA was extracted from 24 biopsies of T-cell lymphomas, 12 biopsies of reactive lymphoid infiltrates, and 2 biopsies of primary cutaneous large B-cell lymphomas. Vγ1–8, Vγ9, Vγ10, Vγ11, and Jγ1/Jγ2 consensus primers were used for TCR-γ gene rearrangement amplification and PCR products were analyzed by nonradioactive SSCP. Monoclonal controls yielded a well-defined banded pattern, whereas all polyclonal T-cell controls showed a reproducible pattern of smears. We detected monoclonality in 20/21 (95%) T-cell lymphoma cases, whereas no dominant T-cell clones were found in any of the reactive lymphoid infiltrates or B-cell lymphomas. Sensitivity of 1–5% was demonstrated by serially diluting Jurkat cells in mononuclear blood cells from normal individuals. We conclude that nonradioactive PCR-SSCP for TCR-γ gene rearrangement analysis is a useful adjunct to routine histological and immunophenotypic methods in the diagnosis of T-cell lymphoproliferative disorders in paraffin-embedded tissue. PMID:9916920

Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues. PMID:26121314

Summary Epidermal growth factor receptor (EGFR) is a member of the erbB tyrosine kinase family reported to be overexpressed in a variety of solid malignancies. Mutations in exons 19 to 21 of the tyrosine kinase domain have been detected in a subset of these tumors and its presence associated with a better response to EGFR inhibitors. Several clinical trials are currently underway to evaluate the performance of such drugs in patients with bladder cancer, but data on EGFR mutation status are limited. The current study assesses EGFR immunohistochemical expression and the presence of mutations in exons 19 and 21 by polymerase chain reaction in 19 bladder urothelial carcinomas from formalin-fixed, paraffin-embedded tissues. Representative paraffinsections were microdissected for DNA extraction using a pinpoint isolation system. Parallel sections were immunostained using a monoclonal anti-EGFR antibody. No mutations in exons 19 and 21 of EGFR were identified in any of the cases. Immunohistochemical EGFR positivity was observed in 14 of 19 cases. In summary, we found EGFR protein expression in 74% of urothelial carcinomas, but we failed to detect EGFR mutations at exons 19 to 21, suggesting that EGFR overexpression is not related to the presence of mutations in the tyrosine kinase domain of the gene. Mutation analysis of EGFR exons 19 and 21 is feasible in microdissected paraffinsections from archival tissues. Immunohistochemical expression of EGFR may not be useful to predict therapeutic response to EGFR inhibitors in patients with urothelial carcinomas. To explain EGFR immunohistochemical overexpression, other mechanisms besides mutations in the EGFR kinase domain should be investigated in future studies. PMID:22406363

Nuclear debris may significantly interfere with the analysis of S-phase fraction (SPF) from paraffin-embedded tumors. We used a background subtraction algorithm to compensate for the effects of slicing of tumor cell nuclei during preparation of paraffin-embedded specimens. DNA histograms were analyzed from 88 node-negative breast and from 78 prostatic carcinomas. Median SPFs corrected for nuclear slicing were lower than uncorrected ones in both breast cancer (7.6% vs. 5.7%) and prostate cancer (6.7% vs. 4.2%). The median SPF value in each group was used as a cut-off point in survival studies. As compared with the uncorrected SPFs, corrected SPF levels resulted in a more significant survival difference between breast cancer patients with above and below median SPF (p = 0.0014 vs. p = 0.014) and in a higher relative risk (RR) of death (4.5 vs. 3.1). The same was true for prostate cancer survival (p less than 0.0001 vs. p = 0.002) and RR (5.3 vs. 3.1). Compared with the exponential background subtraction method, the sliced nuclei correction was more reproducible and could be applied in all evaluable histograms without the risk of overcompensation. In conclusion, our results support the use of background correction with the sliced nuclei model in DNA flow cytometric studies of archival tissues. PMID:1935457

In captive penguins, avian malaria due to Plasmodium parasites is a well-recognized disease problem as these protozoa may cause severe losses among valuable collections of zoo birds. In blood films from naturally infected birds, identification and differentiation of malaria parasites based on morphological criteria are difficult because parasitaemia is frequently light and blood stages, which are necessary for identification of parasites, are often absent. Post-mortem diagnosis by histological examination of tissue samples is sometimes inconclusive due to the difficulties in differentiating protozoal tissue stages from fragmented nuclei in necrotic tissue. The diagnosis of avian malaria would be facilitated by a technique with the ability to specifically identify developmental stages of Plasmodium in tissue samples. Thus, a chromogenic in-situ hybridization (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 18S rRNA, was developed for the detection of Plasmodium parasites in paraffin wax-embedded tissues. This method was validated in comparison with traditional techniques (histology, polymerase chain reaction), on various tissues from 48 captive penguins that died at the zoological garden Schönbrunn, Vienna, Austria. Meronts of Plasmodium gave clear signals and were easily identified using ISH. Potential cross-reactivity of the probe was ruled out by the negative outcome of the ISH against a number of protozoa and fungi. Thus, ISH proved to be a powerful, specific and sensitive tool for unambiguous detection of Plasmodium parasites in paraffin wax-embedded tissue samples. PMID:21711191

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases. PMID:26066790

In the present study a highly species-specific oligonucleotide sequence of Tritrichomonas foetus 18S rRNA was used to design an antisense probe for identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens by means of fluorescence in situ hybridization (FISH). Using archival histological specimens from several species with light microscopic evidence of intestinal trichomoniasis, and under optimized hybridization conditions, the probe positively identified trichomonads in colonic specimens from piglets and a kitten with PCR-confirmed T. foetus infection. Neither positive hybridization of the probe or PCR amplification of T. foetus DNA was observed in histological specimens from hamster (Tritrichomonas muris), turkey, nor mouse (Entamoeba muris) intestinal protozoal infections. Sequence-specific binding of the probe was further verified by successfully out-competing the hybridization with 10 x molar excess unlabeled probe and failure of a labeled sense probe to hybridize. The FISH assay described here enables simultaneous location and molecular identification of T. foetus in formalin-fixed and paraffin-embedded histological specimens of intestinal trichomoniasis. The methods employed are likely to also be applicable to probes designed for specific recognition of other trichomonad species, especially in mammalian tissue where red blood cell auto-fluorescence can be easily differentiated from the hybridization signal of trichomonads. PMID:20447769

The diagnosis of canine leishmaniosis (CanL) is currently predominantly achieved by cytological or histological identification of amastigotes in biopsy samples, demonstration of specific anti-Leishmania antibodies and PCR-based approaches. All these methods have the advantage of being sensitive and more or less specific; nevertheless, most of them also have disadvantages. A chromogenic in situ hybridisation (ISH) procedure with a digoxigenin-labelled probe, targeting a fragment of the 5.8S rRNA was developed for the detection of all species of Leishmania parasites in routinely paraffin wax-embedded canine tissues. This method was validated in comparison with traditional techniques (histology, PCR), on various tissues from three dogs with histological changes consistent with a florid leishmaniosis. Amastigote forms of Leishmania gave clear signals and were easily identified using ISH. Various tissues from 10 additional dogs with clinical suspicion or/and a positive serological test but without histological presence of amastigotes did not show any ISH signals. Potential cross-reactivity of the probe was ruled out by negative outcome of the ISH against selected protozoa (including the related Trypanosoma cruzi) and fungi. Thus, ISH proved to be a powerful tool for unambiguous detection of Leishmania parasites in paraffin wax-embedded tissues. PMID:21921059

Cystic echinococcosis (CE), caused by the larval stage of Echinococcus granulosus, presents an important medical and veterinary problem globally, including that in Iran. Different genotypes of E. granulosus have been reported from human isolates worldwide. This study identifies the genotype of the parasite responsible for human hydatidosis in three provinces of Iran using formalin-fixed paraffin-embedded tissue samples. In this study, 200 formalin-fixed paraffin-embedded tissue samples from human CE cases were collected from Alborz, Tehran, and Kerman provinces. Polymerase chain reaction amplification and sequencing of the partial mitochondrial cytochrome c oxidase subunit 1 gene were performed for genetic characterization of the samples. Phylogenetic analysis of the isolates from this study and reference sequences of different genotypes was done using a maximum likelihood method. In total, 54.4%, 0.8%, 1%, and 40.8% of the samples were identified as the G1, G2, G3, and G6 genotypes, respectively. The findings of the current study confirm the G1 genotype (sheep strain) to be the most prevalent genotype involved in human CE cases in Iran and indicates the high prevalence of the G6 genotype with a high infectivity for humans. Furthermore, this study illustrates the first documented human CE case in Iran infected with the G2 genotype. PMID:25535316

Dysregulated proteolysis represents a hallmark of numerous diseases. In recent years, increasing number of studies has begun looking at the protein termini in hope to unveil the physiological and pathological functions of proteases in clinical research. However, the availability of cryopreserved tissue specimens is often limited. Alternatively, formalin-fixed, paraffin-embedded (FFPE) tissues offer an invaluable resource for clinical research. Pathologically relevant tissues are often stored as FFPE, which represent the most abundant resource of archived human specimens. In this study, we established a robust workflow to investigate native and protease-generated protein N termini from FFPE specimens. We demonstrate comparable N-terminomes of cryopreserved and formalin-fixed tissue, thereby showing that formalin fixation/paraffinembedment does not proteolytically damage proteins. Accordingly, FFPE specimens are fully amenable to N-terminal analysis. Moreover, we demonstrate feasibility of FFPE-degradomics in a quantitative N-terminomic study of FFPE liver specimens from cathepsin L deficient or wild-type mice. Using a machine learning approach in combination with the previously determined cathepsin L specificity, we successfully identify a number of potential cathepsin L cleavage sites. Our study establishes FFPE specimens as a valuable alternative to cryopreserved tissues for degradomic studies. PMID:27087653

Rust fungi infect a wide range of plant species making them of particular interest to plant pathologists. In order to study the interactions between these important pathogenic fungi and their host plants it is useful to be able to differentiate fungal tissue from plant tissue. This can be accomplish...

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

Background Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffinembedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods. Results All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations. Conclusions Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS. PMID:23590575

Whilst next generation sequencing can report point mutations in fixed tissue tumour samples reliably, the accurate determination of copy number is more challenging. The conventional Multiplex Ligation-dependent Probe Amplification (MLPA) assay is an effective tool for measurement of gene dosage, but is restricted to around 50 targets due to size resolution of the MLPA probes. By switching from a size-resolved format, to a sequence-resolved format we developed a scalable, high-throughput, quantitative assay. MLPA-seq is capable of detecting deletions, duplications, and amplifications in as little as 5ng of genomic DNA, including from formalin-fixed paraffin-embedded (FFPE) tumour samples. We show that this method can detect BRCA1, BRCA2, ERBB2 and CCNE1 copy number changes in DNA extracted from snap-frozen and FFPE tumour tissue, with 100% sensitivity and >99.5% specificity. PMID:26569395

The novel HPV Direct Flow CHIP commercial system for Human Papillomavirus (HPV) genotyping is based on rapid PCR and automatic reverse dot blot hybridization to genotype-specific probes, allowing the detection of 36 HPV genotypes. This study examined the performance of HPV Direct Flow CHIP in formalin-fixed paraffin-embedded (FFPE) samples (n= 99). Each sample was analyzed both by Direct PCR, using crude cell extracts without DNA purification, and by conventional PCR, using purified DNA. Pair-wise analysis of the results demonstrated strong concordance between the results obtained with the two protocols, although a slightly higher rate of multiple infections was detected by conventional PCR. In summary, HPV Direct Flow CHIP achieves effective HPV detection from FFPE samples with both Direct PCR and Conventional PCR protocols. PMID:24222806

The molecular mechanism responsible for Ewing’s Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis. PMID:27144561

Purpose. The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic “hotspot” regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. Methods. We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. Results. Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed “true-positive” gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent “false-positive” calls in clinically druggable oncogenes such as PIK3CA. Conclusion. AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent “false-positive” variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making. PMID:24664487

Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors. PMID:24249219

The gallocyanin chromalum stain belongs to the classical DNA-RNA staining techniques in histochemistry. It has some important features for successful object orientated image analysis of whole sections of the human brain. To obtain reproducible staining results with these large sections, the method of Einarson was adapted to image analytical requirements. We discuss staining in a warm staining solution, pH adjustment, and optimal stain composition. The embedding procedure for whole human brains is considered as well. PMID:9554583

Traditional histological protocols in marine fish reproductive laboratories using paraffin as the embedding medium are now increasingly being replaced with protocols using resin instead. These procedures entail different degrees of tissue shrinkage complicating direct comparisons of measurement results across laboratories or articles. In this work we selected ovaries of spawning Mediterranean albacore (Thunnus alalunga) as the subject of our study to address the issue of structural changes, by contrasting values on oocyte recruitment and final batch fecundity given from the same tissue samples in both paraffin and resin. A modern stereological method, the oocyte packing density (OPD) theory, was used supported by initial studies on ovarian tissue sampling and measurement design. Examples of differences in the volume fraction of oocyte stages, free space and connective tissue were found between the embedding media. Mean oocyte diameters were smaller in paraffin than in resin with differences ranging between 0.5% in primary growth and 24.3% in hydration (HYD) stage oocytes. Fresh oocyte measurements showed that oocytes shrank as a consequence of the embedding process, reaching the maximal degree of shrinkage for oocytes in the HYD stage (45.8% in paraffin and 26.5% in resin). In order to assess the effect of oocyte shrinkage on the OPD result, and thereby on relative batch fecundity (Fr), oocyte diameters corrected and uncorrected for shrinkage, were used for estimations. Statistical significant differences were found (P < 0.05) between these two approaches in both embedding media. The average Fr was numerically smaller in paraffin compared to resin (86 ± 61 vs. 106 ± 54 oocytes per gram of body mass (mean ± SD)). For both embedding media statistical significant differences (P < 0.05) were seen between Fr results based on either oocytes in the germinal vesicle migration stage or HYD stage. As a valuable adjunct, the present use of the OPD theory made it possible

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other methods by having the highest correlations of differentially expressed genes (DEGs), and best overlap of pathways, between FRO and FFPE groups. The effect of sample time in formalin (18 h or 3 weeks) on gene expression profiles indicated that test article treatment, not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18 h and 3 week FFPE samples compared with FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of time in paraffin on genomic profiles. Ribo-depletion RNA-seq analysis of 8-, 19-, and 26-year-old control blocks resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, untranslated region, intronic, and ribosomal fractions of the transcriptome. Overall, our results indicate that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples. PMID:26361796

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a potential valuable source of samples for clinical research. Since these specimens are banked in hospital archives, large cohorts of samples can be collected in short periods of time which can all be linked with a patients' clinical history. Therefore, the use of FFPE tissue in protein biomarker discovery studies gains interest. However, despite the growing number of FFPE proteome studies in the literature, there is a lack of a FFPE proteomics standard operating procedure (SOP). One of the challenging steps in the development of such a SOP is the ability to obtain an efficient and repeatable extraction of full length FFPE proteins. In this study, the protein extraction efficiency of eight protein extraction buffers is critically compared with GeLC-MS/MS (1D gel electrophoresis followed by in-gel digestion and LC-MS/MS). The data variation caused by using these extraction buffers was investigated since the variation is a very important aspect when using FFPE tissue as a source for biomarker detection. In addition, a qualitative comparison was made between the protein extraction efficiency and repeatability for FFPE tissue and fresh frozen tissue. PMID:26676081

Coinfections with human immunodeficiency virus (HIV) and infectious agents have been recognized since the early 90s. In the central nervous system (CNS) of HIV+ patients, parasitic protozoans like Toxoplasma gondii have been described as responsible for the space occupying lesions (SOL) developed. However, the involvement of Trypanosoma cruzi is also described but appears to be less frequent in acquired immunodeficiency syndrome (AIDS) and transplant recipients, associated with necrotizing myocarditis and neurological symptoms related to the occurrence of necrotizing pseudotumoral encephalitis (NPE) and meningoencephalitis (NME). The present work aims to present a Venezuelan case of NME associated with the coinfection of HIV and a T. cruzi-like trypanosomatid as well as its evolution and diagnosis by histopathological techniques, electron microscopy, and PCR methods using formalin-fixed- (FF-) and paraffin-embedded- (PE-) tissues. Postmortem cytological studies of leptomeninges imprints reveal the presence of trypomastigotes of Trypanosoma sp. Histopathological and electron microscopy studies allowed us to identify an amastigote stage and to reject the involvement of other opportunistic microorganisms as the etiological agent of the SOL. The definitive confirmation of T. cruzi as the etiological agent was achieved by PCR suggesting that the NME by T. cruzi was due to a reactivation of Chagas' disease. PMID:25763312

Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are the most common methods that are used to quantify HER-2 gene and protein levels, respectively, in human breast cancer. However, due to bad sample quality, some samples are unable to be subjected to a FISH assay. We evaluated 71 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by quantitative real-time polymerase chain reaction (qPCR), IHC, and FISH. We also performed qPCR and FISH assays on delayed formalin-fixed (DDF) samples. The qPCR results were in complete concordance with the results of IHC and FISH. In regards to the DDF samples, the HER-2 fluorescent signal seemed decayed compared with that of the DDF samples after 1 h. However, the qPCR method still works well up to 12 hours. Our results indicated that qPCR was obviously superior to FISH in cases that were not fixed in a reasonable amount of time. However, qPCR can be an alternative method by which to perform HER2 amplification assays in breast cancer. PMID:26617766

Proteomic analysis of samples isolated by laser capture microdissection from clinical specimens requires sample preparation and fractionation methods suitable for small amounts of protein. Here we describe a streamlined filter-aided sample preparation (FASP) workflow that allows efficient analysis of lysates from low numbers of cells. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields in the low to submicrogram range. In a single LC-MS/MS run, analyses of 500, 1000, and 3000 cells allowed identification of 905, 1536, and 2055 proteins, respectively. Incorporation of an additional SAX fractionation step at somewhat higher amounts enabled the analysis of formalin fixed and paraffinembedded human tissues prepared by LCM to a depth of 3600-4400 proteins per single experiment. We applied this workflow to compare archival neoplastic and matched normal colonic mucosa cancer specimens for three patients. Label-free quantification of more than 6000 proteins verified this technology through the differential expression of 30 known colon cancer markers. These included Carcino-Embryonic Antigen (CEA), the most widely used colon cancer marker, complement decay accelerating factor (DAF, CD55) and Metastasis-associated in colon cancer protein 1 (MACC1). Concordant with literature knowledge, mucin 1 was overexpressed and mucin 2 underexpressed in all three patients. These results show that FASP is suitable for the low level analysis of microdissected tissue and that it has the potential for exploration of clinical samples for biomarker and drug target discovery. PMID:21526778

Breast cancer is the most common malignancy among females in the world. Age and familial history are the major risk factors for the development of this disease in Iran. Mutations of BRCA1 and BRCA2 genes are associated with a greatly increased risk for development of familial breast cancer. Frequency of BRCA mutations was identified in familial breast cancers (FBC) and non-familial breast cancers (NFBC) by molecular genetics, morphological and Immunohistochemical methods. Thirty forth formalin-fixed, paraffin-embedded breast tissue tumors were analyzed from 16 patients with FBC and 18 patients with NFBC. Three 5382insC mutations detected by multiplex PCR in 16 familial breast cancers. Immunohistochemical method was used to detect estrogen receptor (ER) and progesterona receptor (PR) and TP53. Comparison of ER, PR and TP53 exhibited high difference (P < 0.0001) in familial breast cancers and non-familial breast cancers. Our results demonstrated that 5382insC mutation, ER, PR, TP53, mitotic activity, polymorphism, necrosis and tubules can serve as the major risk factors for the development of FBC. PMID:18630122

Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity. PMID:26864208

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF. PMID:26974561

Infections caused by Histoplasma capsulatum are found most often in endemic regions of North, Central, and South America. H. capsulatum has been divided into eight geographic clades by multi-locus sequence typing (MLST). Recently, one isolate and five formalin-fixed paraffin-embedded (FFPE) tissue samples were received from six of 15 suspected cases of histoplasmosis in cats residing in areas not known to be endemic for H. capsulatum. Polymerase chain reaction (PCR) amplification and sequence analysis of the rDNA ITS-2 region confirmed the diagnosis of H. capsulatum. Since these cases were not, as noted, from the accepted endemic areas, it was of interest to understand the molecular epidemiology of these isolates. Results of molecular analysis indicated that the H. capsulatum recovered from the cats were most closely related to the North American-1 clade, but clustered separately outside this clade, suggesting that the H. capsulatum infecting the animals may represent a separate clade or phylogenetic species. This study also demonstrated the utility of obtaining valuable molecular subtype data directly from archived FFPE tissue blocks, particularly when a fungus culture was not performed or is otherwise unavailable. PMID:23072593

Since Helicobacter spp. DNA was identified in liver tissue resected from patients with hepatocelullar carcinoma (HCC), researchers have suggested a role of this bacterium in hepatic carcinogenesis. Archives of formalin-fixed, paraffin-embedded (FFPE) tissues represent an extraordinary source for clinical studies providing many advantages. However, DNA extraction from FFPE tissues is laborious, time-consuming and still remains a challenge. The aim of this study was to evaluate five protocols for DNA extraction from FFPE liver obtained from patients with HCC in order to detect Helicobacter pylori DNA. These methods were: (1) QIAamp FFPE Tissue Kit, (2) QIAamp DNA Mini Kit, (3) Wizard SV Genomic DNA Purification System, (4) RealiaPrep FFPE gDNA Miniprep System and (5) phenol-chloroform. H. pylori detection was performed using 16S rRNA gene amplification by PCR. The highest total amount of DNA was obtained using the phenol-chloroform method. Analyses of 16S rRNA gene amplification did not show statistically significant differences among the methods (p=0.466), although the highest percentage of positive cases (70%) was found in samples extracted with phenol-chloroform. We suggest that of the five methods evaluated, phenol/chloroform is the most suitable for detection of H. pylori in FFPE liver from patients with HCC. PMID:24355442

Gene copy number aberrations (CNAs) represent a major class of cancer-related genomic alterations that drive solid tumors. Comprehensive and sensitive detection of CNAs is challenging because of often low quality and quantity of DNA isolated from the formalin-fixed, paraffin-embedded (FFPE) solid tumor samples. Here, in a clinical molecular diagnostic laboratory, we tested the utility and validated a molecular inversion probe-based (MIP) array to routinely screen for CNAs in solid tumors. Using low-input FFPE DNA, the array detects genome-wide CNAs with a special focus on 900 cancer-related genes. A cohort of 76 solid tumors of various types and tumor cellularity (20% to 100%), and four cancer cell lines were used. These harbored CNAs in clinically important genes (ERBB2, EGFR, FGFR1, KRAS, MYC) as detected by orthogonal techniques like next-generation sequencing or fluorescence in situ hybridization. Results of the MIP array were concordant with results from orthogonal techniques, and also provided additional information regarding the allelic nature of the CNAs. Limit-of-detection and assay reproducibility studies showed a high degree of sensitivity and reproducibility of detection, respectively. FFPE compatibility, ability to detect CNAs with high sensitivity, accuracy, and provide valuable information such as loss of heterozygosity along with relatively short turnaround times makes the MIP array a desirable clinical platform for routine screening of solid tumors in a clinical laboratory. PMID:27392636

Readily accessible formalin-fixed paraffinembedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. PMID:26551081

Identification of the etiologic agent responsible for sinus fungal ball (SFB) is rarely obtained due to either the culture of patient specimens not being ordered or if cultures were inoculated they proved to be negative. Obviously, this has a significant impact on the design of appropriate therapeutic strategies. We investigated whether paraffin-embedded (PE) tissues, the only materials often available, were suitable for the correct identification of the responsible fungi. We obtained PE tissues of SFB from 16 different patients who had risk factors for invasive fungal infections. DNA was extracted using an automated extractor and the internal transcribed spacer (ITS) sequenced following amplification with two sets of primers designed to amplify >300 bp fragments. This was attempted in parallel with a real-time quantitative PCR assay targeting Aspergillus spp. mitochondrial DNA designed to amplify <150 bp fragments. ITS sequencing succeeded in appropriately identifying the etiologic agents in 10 of the 16 samples (nine Aspergillus fumigatus, one Lewia spp.). In contrast, the <150 bp PCR assay amplified all specimens correctly except the one involving Lewia spp. If fungal identification is warranted to understand the pathophysiology of SFB and guide clinicians, we cannot rely only on ITS sequencing of the DNA obtained from PE tissues. The main reason is probably due to the fact that formalin prevents amplification of long DNA fragments and consequently, frozen or fresh tissues should be employed. PMID:20950222

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging is a rapidly evolving field in which mass spectrometry techniques are applied directly on tissues to characterize the spatial distribution of various molecules such as lipids, protein/peptides, and recently also N-glycans. Glycans are involved in many biological processes and several glycan changes have been associated with different kinds of cancer, making them an interesting target group to study. An important analytical challenge for the study of glycans by MALDI mass spectrometry is the labile character of sialic acid groups which are prone to in-source/postsource decay, thereby biasing the recorded glycan profile. We therefore developed a linkage-specific sialic acid derivatization by dimethylamidation and subsequent amidation and transferred this onto formalin-fixed paraffin-embedded (FFPE) tissues for MALDI imaging of N-glycans. Our results show (i) the successful stabilization of sialic acids in a linkage specific manner, thereby not only increasing the detection range, but also adding biological meaning, (ii) that no noticeable lateral diffusion is induced during to sample preparation, (iii) the potential of mass spectrometry imaging to spatially characterize the N-glycan expression within heterogeneous tissues. PMID:27145236

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective. PMID:25277813

Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer. PMID:27187374

Tumors whose primary site is challenging to diagnose represent a considerable proportion of new cancer cases. We present validation study results for a gene expression-based diagnostic test (the Pathwork Tissue of Origin Test) that aids in determining the tissue of origin using formalin-fixed, paraffin-embedded (FFPE) specimens. Microarray data files were generated for 462 metastatic, poorly differentiated, or undifferentiated FFPE tumor specimens, all of which had a reference diagnosis. The reference diagnoses were masked, and the microarray data files were analyzed using a 2000-gene classification model. The algorithm quantifies the similarity between RNA expression patterns of the study specimens and the 15 tissues on the test panel. Among the 462 specimens, overall agreement with the reference diagnosis was 89% (95% CI, 85% to 91%). In addition to the positive test results (ie, rule-ins), an average of 12 tissues for each specimen could be ruled out with >99% probability. The large size of this study increases confidence in the test results. A multisite reproducibility study showed 89.3% concordance between laboratories. The Tissue of Origin Test makes the benefits of microarray-based gene expression tests for tumor diagnosis available for use with the most common type of histology specimen (ie, FFPE). PMID:21227394

Background The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Experimental design DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. Results DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. Conclusions These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth. PMID:25097466

Background Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffinembedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making. Method We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering. Results Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data. Conclusion Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA. PMID:26919633

Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer. PMID:27187374

The differential diagnosis between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) can be challenging. BL has been reported to express less BCL2 than DLBCL, but this issue has not been analysed systematically. BL expressing BCL2 can be considered to be MYC/BCL2 co-expressors, a feature that is associated with poorer outcome in DLBCL but that has not been correlated with outcome in BL so far. We analysed the expression of BCL2 in 150 cases of conventionally diagnosed BL using two different BCL2 antibodies. BCL2 expression was detected in 23% of the cases, though the expression varied in intensity and number of positive cells. We did not detect any relevant differences in clinical presentation and outcome between BCL2-positive and BCL2-negative BL in a subgroup of 43 cases for which detailed clinical data were available. An independent cohort of 17 BL with expression of BCL2 were analysed molecularly, with 13 of 17 cases classified as molecularly defined BL (Burkitt Lymphoma) using gene expression profiling on formalin-fixed paraffin-embedded tissues. The four lymphomas diagnosed molecularly as intermediates did not differ in clinical presentation and outcome from molecularly defined BL. PMID:26218299

Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8011782

We examined 81 cases of Hodgkin's disease for evidence of the t(14;18) translocation, using the polymerase chain reaction assay on lysates of formalin-fixed, paraffin-embedded tissue. Seven of 74 amplifiable cases (9%) were positive for the translocation, which involves the bcl-2 oncogene and the immunoglobulin heavy chain gene. Two of these cases were sequenced and the breakpoints had the same pattern found in follicular lymphoma. The nuclei from one of the cases were sorted into large and small subpopulations. The t(14;18) signal was more intense in the large nucleus subpopulation, which contained a greater proportion of Reed-Sternberg-like nuclei. These results are consistent with the hypothesis that Reed-Sternberg cells carry the translocation, but they do not exclude the possibility that the translocation is found in cells representing the reactive component of Hodgkin's disease. The results also demonstrate that routinely processed material is suitable for polymerase chain reaction-based analysis of translocations, although the sensitivity is reduced 10- to 100-fold, compared with fresh tissue. Images Figure 1 Figure 2 Figure 3 Figure 5 PMID:8434638

This is the first study from Central America to analyze genetic mutations and histopathological features associated with gastrointestinal stromal tumors (GIST). Mutations found in the tyrosine kinase membrane receptors c-kit and pdgfra are associated with clinical and pathological characteristics of GIST. New drugs that inhibit the expression of these oncogenes at the molecular level substantially improve the quality of life for patients with this tumor. It is therefore essential for patient care in Panama that genetic analysis of GIST tumors continues to develop from the pilot study presented herein into routine clinical use. This study evaluated 39 cases of GIST in Panama, using samples archived at the Instituto Oncológico Nacional from 1994 to 2004. DNA from paraffin‑embedded tumor tissues was isolated and amplified for the exons of c-kit and pdgfra associated with a high frequency of mutations. Direct PCR sequencing of specific exons was performed, and those with different alleles were cloned and re-sequenced. Amino acid sequences were inferred from DNA and aligned to Genbank reference sequences to determine the position and type of mutation. The highest frequency of mutations was found in exon 11 of the c-kit gene (70%). Mutations found in this exon were heterogeneous, while only one type of mutation (p.A502_Y503dup) was observed in c-kit exon 9. Mutations in the pdgfra gene constituted several substitutions, with the deletion p.D842V being observed most frequently. The observed GIST-associated mutations were previously described. Four patients with mutations associated with familial GIST were also found. The majority (66%) of patients with mutations in exon 11 (residues 550-591) were considered to be at high risk and 75% of patients with mutations specifically within residues 556-560 (exon 11) were considered to have high-risk GIST. This is the first molecular study of GIST in Central America. It was performed to gain a better understanding of the cancer

Overexpression of the ATP-dependent drug efflux pump ABCG2 is a major molecular mechanism of multidrug resistance in cancer and might be a predictive biomarker for drug response. Contradictory results have been reported for immunohistochemical studies of ABCG2 protein expression in colorectal cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we established scoring guidelines for ABCG2 expression based on the clinically used guidelines for HER2 immunohistochemistry assessment in gastric cancer. The guidelines provide a semi-quantitative measure of the basolateral membrane staining of ABCG2 and disregard the apical membrane staining and the cytoplasmic signal. Intra-tumor heterogeneity in ABCG2 immunoreactivity was observed; however, statistical analyses of tissue microarrays (TMAs) and the corresponding whole sections from primary tumors of 57 metastatic CRC patients revealed a strong positive correlation between maximum TMA scores and whole sections, especially when more than one core was used. In conclusion, here, we provide validated results to guide future studies on the associations between ABCG2 immunoreactivity in tumor cells and the benefits of chemotherapeutic treatment in patients with CRC. PMID:27257141

Background Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column. Methods Three DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test. Results The combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods. Conclusions Using H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However, one method (TaKaRa) could extract fungal DNA from 69 of the 74 FFPE tissues from which a housekeeping gene could be amplified and was also cost-effective, with a nonlaborious protocol. Factors such as sensitivity, cost, and labor will help guide the selection of the most appropriate method for the needs of each laboratory. PMID:20392915

PCNA is a 36-KD proliferating cell nuclear antigen associated with the cell cycle. The immunocytochemical detection of PCNA represents a useful tool for the study of tumor proliferation activity. This study documents the detection of PCNA, using antibody PC 10 in formalin-fixed, paraffin-embedded tissue, and correlates the proliferative activity of the non-Hodgkin's lymphomas (NHL) with histological grading assessed by the International Working Formulation (WF) and Kiel classification. In 92 cases of NHLs we found a strong correlation between the PCNA index and lymphoma grading. Statistically significant differences were also found between the proliferative index (PI) in low and high grade lymphomas according to the Kiel classification (t = 9.519; p < 0.001) and between low, intermediate and high grade lymphomas according to the WF classification (F = 79.01; p < 0.001). In the Kiel classification the mean of low grade lymphomas was 39.5% and of high grade 75.7%. In the WF the average of low grade lymphomas was 29.7%, intermediate 53.1% and high 75.1%. Although the differences among the groups had been significant, we found variations inside each histological subgroup in both classifications. The intermediate lymphomas were the most heterogeneous group, with PI inside the same histologic subtypes coincident with low and high grade lymphomas. Since PCNA may be used as a marker of cell proliferation in clinical studies to estimate the biological aggressiveness of lymphomas, its determination in intermediate grade NHL could be very useful to evaluate individual cases in this group and determine prognosis and probably the appropriate therapy. PMID:8714262

Phage display is an effective method for the generation of target-specific human antibodies. Standard phage display panning use purified proteins, antigen-transfected cells or tumor cell lines as target structure to generate specific antibodies. However, recombinant proteins can be difficult to express and purify in their native conformation and suitable cell lines are not always available. Additionally the antigen expression profile may change during cultivation and thus differ from the malignant cells in patient. Here we describe a method for the selection of specific antibodies from phage display libraries by panning against formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides, using small cell lung cancer (SCLC) as a case study. The human Tomlinson single-chain variable fragment (scFv) phage libraries I and J were panned against SCLC FFPE tissue slides for positive selection and healthy lung tissue for subtraction. The specificity of the selected scFv antibodies was confirmed in vitro by ELISA on immobilized SCLC cell membranes, by flow cytometry using the SCLC cell lines NCI-H69, NCI-H82 and DMS 273, and ex vivo against tissue microarrays containing 35 different SCLC samples and 20 types of normal organs. We monitored the internalization of three selected scFv antibodies and fused them with Pseudomonas exotoxin A (ETA') to produce immunotoxins whose cytotoxicity was confirmed by cell viability and apoptosis assays on different SCLC cell lines, achieving IC50 values of up to 23nM. The selection of SCLC-specific scFv antibodies by panning against FFPE tissue slides circumvents the challenges of using purified antigens or cell lines for antibody selection. PMID:26045318

Formalin-fixed paraffin-embedded (FFPE) tissue specimens represent a valuable and abundant resource of pathologic material for various biomedical studies. In the present study, we report the application of high-resolution inductively coupled mass-spectrometry (ICP-MS) for quantification of Fe, Zn, Se and Cd in FFPE prostate tissue. These elements have a possible role in the development of prostate diseases: while Zn and Se are needed for a healthy prostate, Cd shows multiple toxic and carcinogenic effects. Excessive accumulation of Fe induces the production of highly reactive hydroxyl radical species, which may play a role in cancer etiopathogenesis. To assess whether the levels of these metals in the FFPE prostate tissue represent their original content, we compared their levels with those in the fresh tissue (on dry weight basis) in samples obtained from 15 patients. We found that in FFPE tissue, the recoveries of Se, Fe, Cd and Zn were progressively decreased, 97??11% (r=0.88), 82??22% (r=0.86), 59??23% (r=0.69) and 24??11% (r=0.38), respectively. Thus, the use of correction factors, determined as k=0.16 for Se, k=0.20 for Fe, k=0.27 for Cd and k=0.67 for Zn, is required to estimate the retrospective levels of these elements in the parental non-processed fresh (wet) prostate tissue. The technique used in this study enables the analysis of archival FFPE prostate tissue for the concentrations of Fe, Zn, Se and Cd to study association between the levels of these metals and prostate disease. ?? 2008.

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues has been used in the past to analyze genetic polymorphisms. We evaluated the technical reproducibility of different types of assays for gene polymorphisms using DNA extracted from FFPE material. By using the MassARRAY iPLEX system, we investigated polymorphisms in DPYD (rs1801159 and rs3918290), UMPS (rs1801019), ERCC1 (rs11615), ERCC1 (rs3212986), and ERCC2 (rs13181) in 56 FFPE DNA samples. By using PCR, followed by size-based gel electrophoresis, we also examined TYMS 5′ untranslated region 2R/3R repeats and GSTT1 deletions in 50 FFPE DNA samples and 34 DNAs extracted from fresh-frozen tissues and cell lines. Each polymorphism was analyzed by two independent runs. We found that iPLEX biomarker assays measuring single-nucleotide polymorphisms provided consistent concordant results. However, by using FFPE DNA, size-based PCR biomarkers (GSTT1 and TYMS 5′ untranslated region) were discrepant in 32.7% (16/49, with exact 95% CI, 19.9%–47.5%; exact binomial confidence limit test) and 4.2% (2/48, with exact 95% CI, 0.5%–14.3%) of cases, respectively, whereas no discrepancies were observed using intact genomic DNA. Our findings suggest that DNA from FFPE material can be used to reliably test single-nucleotide polymorphisms. However, results based on size-based PCR biomarkers, and particularly GSTT1 deletions, using FFPE DNA need to be interpreted with caution. Independent repeated assays should be performed on all cases to assess potential discrepancies. PMID:25746798

Background: Dysregulation of miR-675 has been found in a variety of solid tumors. MiR-675 has been suggested as having both oncogenic and tumor suppression properties in cancer. However, there is no evidence whether miR-675 is involved in breast cancer. The objective of this study was to evaluate the expression status of miR-675 and its clinical relevance in breast cancer patients. Methods: The expression level of miR-675 was detected in 100 breast cancer patients and 38 cancer-free controls using real-time quantitative PCR. The clinicopathological characteristics of miR-675 in breast cancer were also investigated. All statistical analyses were performed using SPSS 20.0. Results: The study showed that miR-675 was significantly up-regulated in breast cancer patients compared with controls (P < 0.01). There was no significant difference in age, lymph nodes stage, ER status and PR status between patients with and without miR-675 over-expression (P > 0.05). The frequency of miR-675 over-expression was higher in the patients of histological grade I-II than in others (50% versus 9%, P = 0.011). The expression level of miR-675 had a high correlation with miR-24/93/98/378 in breast cancer patients. Conclusions: Taken together, our study demonstrated that miR-675 in formalin-fixed paraffin-embedded (FFPE) tissues might serve as a good source for biomarker discovery and breast cancer validation. PMID:26379923

Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffinembedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer. PMID:25961742

Background Differential diagnosis between malignant follicular thyroid cancer (FTC) and benign follicular thyroid adenoma (FTA) is a great challenge for even an experienced pathologist and requires special effort. Molecular markers may potentially support a differential diagnosis between FTC and FTA in postoperative specimens. The purpose of this study was to derive molecular support for differential post-operative diagnosis, in the form of a simple multigene mRNA-based classifier that would differentiate between FTC and FTA tissue samples. Methods A molecular classifier was created based on a combined analysis of two microarray datasets (using 66 thyroid samples). The performance of the classifier was assessed using an independent dataset comprising 71 formalin-fixed paraffin-embedded (FFPE) samples (31 FTC and 40 FTA), which were analysed by quantitative real-time PCR (qPCR). In addition, three other microarray datasets (62 samples) were used to confirm the utility of the classifier. Results Five of 8 genes selected from training datasets (ELMO1, EMCN, ITIH5, KCNAB1, SLCO2A1) were amplified by qPCR in FFPE material from an independent sample set. Three other genes did not amplify in FFPE material, probably due to low abundance. All 5 analysed genes were downregulated in FTC compared to FTA. The sensitivity and specificity of the 5-gene classifier tested on the FFPE dataset were 71% and 72%, respectively. Conclusions The proposed approach could support histopathological examination: 5-gene classifier may aid in molecular discrimination between FTC and FTA in FFPE material. PMID:24099521

Background It is well known that hypertension may cause glomerular damage, but the molecular mechanisms involved are still incompletely understood. Methods In the present study, we used formalin-fixed paraffin-embedded (FFPE) tissue to investigate changes in the glomerular proteome in the non-clipped kidney of two-kidney one-clip (2K1C) hypertensive rats, with special emphasis on the glomerular filtration barrier. 2K1C hypertension was induced in 6-week-old Wistar Hannover rats (n = 6) that were sacrificed 23 weeks later and compared with age-matched sham-operated controls (n = 6). Tissue was stored in FFPE tissue blocks and later prepared on tissue slides for laser microdissection. Glomeruli without severe morphological damage were isolated, and the proteomes were analysed using liquid chromatography–tandem mass spectrometry. Results 2K1C glomeruli showed reduced abundance of proteins important for slit diaphragm complex, such as nephrin, podocin and neph1. The podocyte foot process had a pattern of reduced abundance of transmembrane proteins but unchanged abundances of the podocyte cytoskeletal proteins synaptopodin and α-actinin-4. Lower abundance of important glomerular basement membrane proteins was seen. Possible glomerular markers of damage with increased abundance in 2K1C were transgelin, desmin and acyl-coenzyme A thioesterase 1. Conclusions Microdissection and tandem mass spectrometry could be used to investigate the proteome of isolated glomeruli from FFPE tissue. Glomerular filtration barrier proteins had reduced abundance in the non-clipped kidney of 2K1C hypertensive rats. PMID:25129444

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5+/6+ (BSGP)5+/6+. To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost. PMID:27006646

Sixty-seven formalin-fixed and paraffin-embedded liver biopsies from HBsAg-negative alcoholics without previous blood transfusions or intravenous drug abuse were analyzed for the presence of low-level hepatitis B virus DNA by the polymerase chain reaction. To simplify and standardize the amplification procedure, aliquots of a complete polymerase chain reaction mix were prepared and frozen for storage; random samples were tested prior to analysis of clinical material. Freezing and storage of the aliquots did not affect the activity of Taq polymerase. One large batch of ready-to-use aliquots could thus be used as a standardized polymerase chain reaction kit for all experiments. The suitability of the extracted material for polymerase chain reaction analysis was tested in two ways. First, the absence of nonspecific polymerase chain reaction inhibitors was demonstrated in all samples by amplifying cloned hepatitis B virus DNA in the presence of extracted material. Second, the integrity of the extracted DNA was tested by amplifying a segment of the beta-globin gene. Twenty-three samples were beta-globin DNA positive and thus contained sufficient amounts of nondegraded DNA. These results emphasize the importance of testing both the absence of nonspecific inhibitors and DNA integrity in DNA samples extracted from fixed tissue. Among the 23 beta-globin positive samples, 12 had cirrhosis (52.1%). Two of these samples were hepatitis B virus DNA positive (8.7%); one of these cases had cirrhosis. Thus, even in the absence of common risk factors, the incidence of hepatitis B virus in this alcoholic population was increased compared to the general population. PMID:8071542

Background: E-cadherin (CDH1) plays an important role in cell–cell adhesion of epithelial tissues. Loss of E-cadherin expression can lead to loss of tissue integrity, metastasis, and cancer progression. Also loss of E-cadherin expression might be related to aberrant promoter methylation of the CDH1 gene. Many studies have been performed on CDH1 promoter methylation, especially in breast cancer. Although most of the studies have used qualitative methods for methylation analysis, this study is designed to quantitatively investigate CDH1 promoter methylation in breast cancer and its correlation with patients’ clinicopathological features. Materials and Methods: Using differential high resolution melting analysis (D-HRMA), the methylation level of the CDH1 gene promoter was quantified in 98 breast cancer formalin-fixed paraffin-embedded (FFPE) tissues and also 10 fresh frozen normal breast tissues. Results: All samples were detected to be methylated at the CDH1 promoter region. About 74.5% of the breast cancer samples were hypermethylated with an average methylation level of around 60%, while 25.5% of the patients were methylated with the mean methylation level of about 33%, and 90% of the normal samples had a mean methylation level of about 18%. Statistical analyses represented a significant correlation between CDH1 promoter methylation and cancer progression hallmarks, such as, clinical stage, nodal involvement, tumor size, and histological grade. Conclusion: In summary, quantitation of CDH1 promoter methylation can serve as a diagnostic and prognostic tool in breast cancer. Also D-HRMA can be used as a fast and reliable method for quantitation of promoter methylation. PMID:27308263

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffinembedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels ofc-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens. PMID:21283737

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffinembedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels of c-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens. PMID:21283737

Triple negative breast cancer (TNBC), defined by the lack of expression of the estrogen receptor, progesterone receptor and human epidermal receptor 2, is an aggressive form of breast cancer that is more prevalent in certain populations, in particular in low- and middle-income regions. The detailed molecular features of TNBC in these regions remain unexplored as samples are mostly accessible as formalin-fixed paraffinembedded (FFPE) archived tissues, a challenging material for advanced genomic and transcriptomic studies. Using dedicated reagents and analysis pipelines, we performed whole exome sequencing and miRNA and mRNA profiling of 12 FFPE tumor tissues collected from pathological archives in Mexico. Sequencing analyses of the tumor tissues and their blood pairs identified TP53 and RB1 genes as the most frequently mutated genes, with a somatic mutation load of 1.7 mutations/exome Mb on average. Transcriptional analyses revealed an overexpression of growth-promoting signals (EGFR, PDGFR, VEGF, PIK3CA, FOXM1), a repression of cell cycle control pathways (TP53, RB1), a deregulation of DNA-repair pathways, and alterations in epigenetic modifiers through miRNA:mRNA network de-regulation. The molecular programs identified were typical of those described in basal-like tumors in other populations. This work demonstrates the feasibility of using archived clinical samples for advanced integrated genomics analyses. It thus opens up opportunities for investigating molecular features of tumors from regions where only FFPE tissues are available, allowing retrospective studies on the search for treatment strategies or on the exploration of the geographic diversity of breast cancer. PMID:25961742

HIV-1 infection of tonsils takes place when virus spreads systemically, and may occur when tonsillar tissue serves as the initial portal of HIV-1 entry. The HIV replication cycle includes the production of regulatory and accessory gene mRNAs, produced by splicing of genomic mRNA, that are hallmarks of de novo virus production. We sought to demonstrate, for the first time, the presence of multiply spliced viral RNA transcripts in archival tissue as a marker for active virus replication. Further, amplified cDNA sequences from unspliced pol gene mRNA were used to define the genetic subtype of HIV-1 within these tissues. RNA was extracted from surgical pathological, formalin-fixed, paraffin-embedded specimens, and RT-PCR was performed with primers for unspliced and multiply spliced HIV-1 transcripts. Amplification products were analyzed by agarose gel electrophoresis and their specificity was confirmed by sequencing and Southern blot hybridization. Unspliced HIV-1 pol transcripts yielded cDNA amplicons of 184 base pairs (bp) that were cloned and sequenced. Phylogenetic analysis revealed these sequences to be of HIV-1 subtype B. Multiply spliced transcripts specific for the tat/rev (173 bp), tat (268 bp), and tat/rev/nef (146 bp) regulatory gene mRNAs could be demonstrated in all cases. These results support the demonstration of active replication of HIV-1 in archival tonsillar tissues previously shown by p24 antigen staining. They also show the feasibility of performing molecular epidemiologic studies on HIV-1 cDNA sequences from archived pathologic specimens. PMID:12167270

Available clinical human papilloma virus (HPV) diagnostics for head and neck cancer have limited sensitivity and/or fail to define the HPV genotype. Common HPV genotyping assays are costly and labor intensive. We sought to develop a next-generation sequencing (NGS)-based HPV genotyping assay that was sensitive enough to work on formalin-fixed paraffin-embedded (FFPE) samples. We developed an ion torrent NGS HPV genotyping assay using barcoded HPV PCR broad-spectrum general primers 5(+)/6(+) (BSGP)5(+)/6(+). To validate genotype specificity and use in archived clinical FFPE tumor samples, we compared NGS HPV genotyping at 2 sequencing centers with typing by Roche Linear Array assay in 42 oropharyngeal and cervical cancer specimens representing 10 HPV genotypes, as well as HPV-negative cases. To demonstrate the detection of a broad range of HPV genotypes, we genotyped a cohort of 266 cervical cancers. A comparison of NGS genotyping of FFPE cancer specimens with genotyping by Linear Array showed concordant results in 34/37 samples (92%) at sequencing site 1 and 39/42 samples (93%) at sequencing site 2. Concordance between sites was 92%. Designed for use with 10 ng genomic DNA, the assay detected HPV using as little as 1.25 ng FFPE-derived genomic DNA. In 266 cervical cancer specimens, the NGS assay identified 20 different HPV genotypes, including all 13 carcinogenic genotypes. This novel NGS assay provides a sensitive and specific high-throughput method to detect and genotype HPV in a range of clinical specimens derived from FFPE with low per-sample cost. PMID:27006646

Early diagnosis and curative resection are the predominant factors associated with increased survival in patients with gastric cancer. However, most gastric cancer cases are still diagnosed at later stages. Since most pathologic specimens are archived as FFPE samples, the ability to use them to generate expression profiles can greatly improve cancer biomarker discovery. We sought to uncover new biomarkers for stomach preneoplastic metaplasias and neoplastic lesions by generating proteome profiles using FFPE samples. We combined peptide isoelectric focusing and liquid chromatography–tandem mass spectrometry analysis to generate proteomic profiles from FFPE samples of intestinal-type gastric cancer, metaplasia, and normal mucosa. The expression patterns of selected proteins were analyzed by immunostaining first in single tissue sections from normal stomach, metaplasia, and gastric cancer and later in larger tissue array cohorts. We detected 60 proteins up-regulated and 87 proteins down-regulated during the progression from normal mucosa to metaplasia to gastric cancer. Two of the up-regulated proteins, LTF and DMBT1, were validated as specific markers for spasmolytic polypeptide–expressing metaplasia and intestinal metaplasia, respectively. In cancers, significantly lower levels of DMBT1 or LTF correlated with more advanced disease and worse prognosis. Thus, proteomic profiling using FFPE samples has led to the identification of two novel markers for stomach metaplasias and gastric cancer prognosis. PMID:22944598

Hepatitis B antigen (HBAg) has been demonstrated in conventional formalin-fixed paraffin-embedded liver tissue by peroxidase and fluorescent immunostaining as well as by orcein. Complete locational and morphologic identity is seen between material stained by specific immunologic methods and by orcein. The antigen is restricted to the cytoplasm and is generally observed in the hepatocyte; it is present in three morphologic forms. Certain morphologic forms can even be identified in hematoxylin and eosin-stained tissue. Results of immunostaining procedures indicate that the antigen demonstrated in this study consists entirely of surface coat of hepatitis B virus (HBsAg). This seems to be the only component revealed by orcein staining. The latter is considered to be a good marker of the surface antigen and to have certain advantages over immunostaining. It is suggested that suitability of conventional paraffinsections for the detection of HBAg has wide and important implications. Images Figures 1-5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:55076

Immunocompromised patients who develop invasive filamentous mycotic infections can be efficiently treated if rapid identification of the causative fungus is obtained. We report a case of fatal necrotic pneumonia caused by combined pulmonary invasive mucormycosis and aspergillosis in a 66 year-old renal transplant recipient. Aspergillus was first identified during the course of the disease by cytological examination and culture (A. fumigatus) of bronchoalveolar fluid. Hyphae of Mucorales (Rhizopus microsporus) were subsequently identified by culture of a tissue specimen taken from the left inferior pulmonary lobe, which was surgically resected two days before the patient died. Histological analysis of the lung parenchyma showed the association of two different filamentous mycoses for which the morphological features were evocative of aspergillosis and mucormycosis. However, the definitive identification of the associative infection was made by polymerase chain reaction (PCR) performed on deparaffinized tissue sections using specific primers for aspergillosis and mucormycosis. This case demonstrates that discrepancies between histological, cytological and mycological analyses can occur in cases of combined mycotic infection. In this regard, it shows that PCR on selected paraffin blocks is a very powerful method for making or confirming the association of different filamentous mycoses and that this method should be made available to pathology laboratories. PMID:20205795

Background: Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT–PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel. Methods: The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data. Results: Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearson's R2=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R2=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes. Conclusion: We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data

Cervical Papanicolaou smears and paraffinsections of biopsy specimens obtained from women attending dysplasia clinics were examined for viral DNA sequences by in situ hybridization technique using TVS-labeled cloned recombinant DNA probes of human papillomavirus (HPV) types 6, 11, and 16. These and one unrelated DNA probe complementary to measles virus RNA were labeled by nick translation using either one or two TVS-labeled nucleotides. Paraffinsections and cervical smears were collected on pretreated slides, hybridized with the probes under stringent or nonstringent conditions for 50 h, and autoradiographed. Additional cervical specimens from the same women were examined for the presence of genus-specific papillomavirus capsid antigen by the immunoperoxidase technique. Preliminary results may be summarized as follows. The infecting virus could be identified in smears as well as in sections. Viral DNA sequences were detected only when there were condylomatous cells in the specimen and in only a proportion of the condylomatous cells. Even under stringent conditions, some specimens reacted with both HPV-6 and HPV-11. In some instances, the cells did not hybridize with any of the three probes even when duplicate specimens contained frankly condylomatous, capsid antigen-positive cells. In situ hybridization of Papanicolaou smears or of tissue sections is a practical method for diagnosis and follow-up of specific papillomavirus infection using routinely collected material.

Introduction Intraoral mucosa-associated lymphoid tissue (MALT) lymphoma is a rare lymphoma that has a good prognosis if diagnosed correctly and treated in time. Presentation of case A 64-year-old woman was referred to our department with asymptomatic swelling of the left hard palate. Computed tomography and magnetic resonance imaging revealed a mass in the left hard palate. We performed a pre-surgery biopsy; however, it was difficult to differentiate MALT lymphoma from other reactive lymphoproliferative disorders via gross or microscopic examination. Although the lesion was completely excised, histological findings did not allow a definitive diagnosis due to an absence of visible monoclonality. We then performed polymerase chain reaction (PCR) using DNA extracted from formalin-fixed, paraffin-embedded surgical samples. Capillary electrophoresis showed monoclonal peaks of immunoglobulin heavy chain gene rearrangement, thus facilitating a definitive diagnosis of MALT lymphoma. Discussion PCR technique is rapid, accurate, and enables a definitive diagnosis without relying on traditional histological or molecular diagnostic techniques, such as Southern blotting. Conclusion We suggest that, if histological examination is ambiguous or fresh material is insufficient, PCR can be performed using paraffin-embedded materials to definitively diagnose low-grade lymphomas, such as MALT lymphoma. PMID:25841155

We present a retrospective study using four different immunohistochemical markers (PCNA, Ki-67, 486p and p53) on paraffinsections from 104 selected cases with primary superficial transitional cell carcinomas of the bladder (59 cases pTa, 45 cases pT1, 40 cases G1, 64 cases G2). 53 of the 104 patients experienced recurrence of their bladder lesion, while 51 remained free of tumor. The distribution of staging, grading and multifocality was comparable in both groups of patients. Overall, the tumors that recurred had a significantly higher proportion of labeled cells for PCNA (p < or = 0.0001), Ki-67 (p < or = 0.006) and 486p (p < or = 0.0001). The latter antigen proved to be the most reliable marker. A less significant difference in staining pattern was found for p53 (p < or = 0.01). Evaluating the predictive value of the various antibodies separately for the groups with G1 vs. G2 carcinomas and pTa vs. pT1 tumors revealed a lower significance for all antibodies. The technique of immunostaining on paraffinsections facilitates further retrospective studies on archival material. These markers may provide additional information about the probability of recurrence of superficial bladder tumors. But at the moment they should only be utilized in selected cases. PMID:9392055

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffinsections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 PMID:2821078

Background Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs. Methods Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks. Results The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy. Conclusions Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks. PMID:23837016

This work aims to enact a quick and reasonable estimation of breast cancer margin thickness using time of flight analysis of embedded breast cancer tissue. A pulsed terahertz system is used to obtain reflection imaging scans from breast cancer tumors that are formalin-fixed and embedded in paraffin blocks. Time of flight analysis is then used to compare the reflection patterns seen within the block to pathology sections and paraffin-embeddedsections that are taken throughout the depth of the tumor in order to estimate the three-dimensional boundaries of the tumor.

A best evidence topic in cardiothoracic surgery was written according to a structured protocol. The question addressed was 'Are frozen sections of mediastinoscopy samples as effective as formal paraffin assessment of mediastinoscopy samples for a decision on a same-day lobectomy?'. Five papers were found using the reported search that represented the best evidence to answer the clinical question. The authors, journal, date and country of publication, patient group studied, study type, relevant outcomes and results of these papers are tabulated. These studies compared the efficacy and accuracy of frozen sections (FSs) from mediastinal lymph nodes for staging of patients with lung cancer to determine whether a combined procedure can be planned based on these results and to proceed to thoracotomy and lung resection in cases of negative mediastinal nodes diagnosed by FS. These studies unanimously showed that FS of mediastinal nodes are as accurate as permanent section results and definite histology diagnosis with a sensitivity of >94% and specificity of 100% with no false-positive results. They also confirmed that even in benign lung conditions and other malignancies of the mediastinum, the results of FS are compared with the histology of the node. Based on the current reports, a combined procedure (staging mediastinal nodes by FS and planning for thoracotomy or abandoning thoracotomy) is a safe approach to treat non-small-cell lung cancer (NSCLC). From the patients' point of view, this approach is superior to the staged procedure (mediastinoscopy followed by lung resection at a later date based on the histology of mediastinal nodes) due to single hospitalization and anaesthesia, however whether it is cost effective or not is debatable. It is also labour-intensive and operator-dependent. In conclusion, the current evidence in the literature suggests that a combined procedure of mediastinal node FS followed by lung resection can be a safe alternative to a staged

The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America. Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions. The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan. The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated. Tissue samples had many yeast-like organisms in the macrophages. DNA was extracted from paraffin-embedded tissue samples. A nested PCR technique was applied. The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum). Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H. capsulatum var. farciminosum as a heteroecism. PMID:12814889

Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EAC (N = 13); progressors to HGD or EAC (BIM-P) with BIM and a worse diagnosis of HGD or EAC (N = 15); and four BIM-negative samples. No mutations were detected in the BIM-NP (0 of 13) or BIM-negative samples, whereas the BIM-P samples had mutations in 6 (75%) of 8 cases in TP53, APC, and CDKN2A (P = 0.0005), detected in samples with as low as 20% BIM. We found that next-generation sequencing from routine FFPE nonneoplastic Barrett's esophagus samples can detect multiple mutations in minute areas of BIM with high analytical sensitivity. Next-generation sequencing panels for detection of TP53 and possibly combined mutations in other genes, such as APC and CDKN2A, may be useful in the clinical setting to improve dysplasia and cancer surveillance in patients with Barrett's esophagus. PMID:26068095

Barrett's intestinal metaplasia (BIM) may harbor genomic mutations before the histologic appearance of dysplasia and cancer and requires frequent surveillance. We explored next-generation sequencing to detect mutations with the analytical sensitivity required to predict concurrent high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC) in patients with Barrett's esophagus by testing nonneoplastic BIM. Formalin-fixed, paraffin-embedded (FFPE) routine biopsy or endoscopic mucosal resection samples from 32 patients were tested: nonprogressors to HGD or EAC (BIM-NP) with BIM, who never had a diagnosis of dysplasia or EAC (N = 13); progressors to HGD or EAC (BIM-P) with BIM and a worse diagnosis of HGD or EAC (N = 15); and four BIM-negative samples. No mutations were detected in the BIM-NP (0 of 13) or BIM-negative samples, whereas the BIM-P samples had mutations in 6 (75%) of 8 cases in TP53, APC, and CDKN2A (P = 0.0005), detected in samples with as low as 20% BIM. We found that next-generation sequencing from routine FFPE nonneoplastic Barrett's esophagus samples can detect multiple mutations in minute areas of BIM with high analytical sensitivity. Next-generation sequencing panels for detection of TP53 and possibly combined mutations in other genes, such as APC and CDKN2A, may be useful in the clinical setting to improve dysplasia and cancer surveillance in patients with Barrett's esophagus. PMID:26068095

Formalin-fixed paraffin-embedded material from 57 patients in whom curative resection of pancreatic carcinoma had been attempted was analysed by an immunohistochemical procedure to estimate proliferation and p53 protein expression. Using the monoclonal antibody (MAb) MIB-1, which recognizes a Ki-67 epitope, the proliferating cell index (PCI, percentage of immunoreactive tumour nuclei) and proliferating cell area (PCA, percentage of immunoreactive tumour nuclear area) were calculated using an interactive image analysis system and were compared with semiquantitative scoring of stainability. MAb DO-7, which recognizes both wild- and mutant-type p53 protein, was used to assess p53 expression in the same material. MIB-1 stainings were of high quality in 53 tumours. The median PCI was 29.7% (range 0.5-82.1%) and the median PCA was 10.6% (range 0.0-36.5%). There was a close correlation between PCI and PCA (P < 0.0001). PCI and PCA values were in conformity with the semiquantitative scoring (P < 0.0001). The p53 immunohistochemical stainings were successful in 48 tumours and the protein was expressed in 22 (46%). High PCI values (> 45%, n = 14) correlated with shorter survival time (P < 0.01). PCA (P < 0.05) and the expression of p53 protein (P < 0.001) were independent prognostic variables. Images Figure 1 Figure 2 PMID:9218733

The Rocky Mountain Oilfield Testing Center (RMOTC) has recently completed a test of a paraffin removal system developed by the KKG Group utilizing the technology of two Russian scientists, Gennady Katzyn and Boris Koggi. The system consisting of chemical ''sticks'' that generate heat in-situ to melt the paraffin deposits in oilfield tubing. The melted paraffin is then brought to the surface utilizing the naturally flowing energy of the well.

This research investigates the application of additive manufacturing techniques for fabricating hybrid rocket fuel grains composed of porous Acrylonitrile-butadiene-styrene impregnated with paraffin wax. The digitally manufactured ABS substrate provides mechanical support for the paraffin fuel material and serves as an additional fuel component. The embeddedparaffin provides an enhanced fuel regression rate while having no detrimental effect on the thermodynamic burn properties of the fuel grain. Multiple fuel grains with various ABS-to-Paraffin mass ratios were fabricated and burned with nitrous oxide. Analytical predictions for end-to-end motor performance and fuel regression are compared against static test results. Baseline fuel grain regression calculations use an enthalpy balance energy analysis with the material and thermodynamic properties based on the mean paraffin/ABS mass fractions within the fuel grain. In support of these analytical comparisons, a novel method for propagating the fuel port burn surface was developed. In this modeling approach the fuel cross section grid is modeled as an image with white pixels representing the fuel and black pixels representing empty or burned grid cells.

To facilitate a detailed investigation of pinniped lymphoid organs, 30 monoclonal antibodies (mAb) as well as eight polyclonal antibodies (pAb) of different species specificities directed against cell antigens of the hematopoietic system were tested for immunohistochemical cross-reactivity on formalin-fixed, paraffin wax-embedded tissues of harbor seals (Phoca vitulina) and a walrus (Odobenus rosmarus rosmarus). Six monoclonal and eight polyclonal antibodies showed specific immunoreactivities. Lymphocytes were immunolabeled by an anti-CD3 pAb, anti-Foxp3 mAb and anti-CD79 alpha mAb, while plasma cell subpopulations were recognized by anti-IgA pAb, anti-IgG pAb and anti-IgM pAb as well as by anti-kappa- and anti-lambda light chain pAb. Cells of the histiocytic lineage were recognized by lysozyme-, myeloid/histiocyte antigen-, and CD68-specific markers. Furthermore, dendritic cell-like cells were detected by an anti-S100 protein pAb. The MHC class II antigen was labeled on the majority of immune cells of the harbor seal and walrus using a bovine mAb. Mast cells were stained by an anti-mast cell tryptase mAb. Thus, using these antibodies from various species, it is now possible to determine phenotypical changes in lymphoid organs and detect different leukocyte subsets involved in inflammatory responses in archived tissue samples of these pinniped species. PMID:20566219

Optimal recovery of nucleic acids from formalin-fixed paraffin-embedded (FFPE) tissues is highly dependent on a series of pre-extraction steps, mainly related (but not limited) to fixation. The aim of our study was to investigate if the storage period of the FFPE blocks had a significant effect on the isolated DNA. We examined the quantity and purity of the isolated DNA from 83 FFPE blocks, corresponding to malignant thyroid (n=28) and renal (n=55) carcinomas that had been stored in our department for up to eight years. The DNA extraction protocol was based on a precipitation method (MasterPure™ DNA Purification Kit, Epicentre), in accordance to the manufacturer instructions, optimized in our laboratory. A spectrophotometer was used to determine the yield (A260) and purity (A260/A280 ratio) of the isolated DNA. We successfully isolated good DNA quantity and purity from all our study cases (mean concentration: 223.4 ± 104.16 ng/μL; mean A260/A280 ratio: 1.68 ± 0.09). Moreover, no statistically significant differences were observed between tumor blocks stored for 2-3 years and 7-8 years, respectively, both in terms of DNA quantity (p=0.196) and purity (p=0.663). In conclusion, we successfully validated an efficient, reproducible DNA extraction technique that provided a good range of DNA concentrations and purity, regardless the type of tissue (thyroid or kidney). Moreover, we demonstrated that the storage period of the FFPE blocks does not have a significant influence on the DNA quantity and purity. PMID:26429169

Lung cancer is the most common cause of cancer-related deaths worldwide, and metastatic spread of the cancer rather than the primary tumor is the main cause of death. However, the molecular alterations of cancer cells leading to the formation of metastasis are poorly understood. This is partly a result of most solid tumor samples available for retrospective studies being archived as formalin-fixed paraffin-embedded (FFPE) specimens causing the nucleic acids to be highly degraded. Furthermore, stably expressed reference genes for normalization of gene expression data using reverse transcriptase quantitative PCR (RT-qPCR) have not been identified for combined analysis of primary lung tumors and the tissues where to the cancer metastasize. Using an optimized RT-qPCR workflow we have analyzed the expression of 23 candidate reference genes in a total of 54 FFPE specimens derived from primary Non-Small Cell Lung Cancer tumors, brain metastases, and lymph node metastases as well as normal lung, lymph node, and brain tissues. We show that every aspect of the workflow is highly reproducible, and the PUM1, TBP, and IPO8 genes were identified as the most stably expressed reference genes among the candidates, by using the GeNorm and NormFinder software programs. Furthermore, we demonstrate that commonly used reference genes such as ACTB (β-actin), GAPDH, and rRNA18S are less stably expressed in the studied samples. The presented workflow and the identified reference genes may facilitate more reliable gene expression studies in lung cancer using RNA from FFPE tissues. PMID:23643276

The availability of drugs targeting the EGFR/HER/erbB signaling pathway has created a need for diagnostics that accurately predict treatment responses. We have developed and characterized a novel assay to provide sensitive and quantitative measures of HER proteins and homodimers in formalin-fixed, paraffin-embedded (FFPE) cell lines and breast tumor tissues, to test these variables. In the VeraTag assay, HER proteins and homodimers are detected through the release of fluorescent tags conjugated to specific HER antibodies, requiring proximity to a second HER antibody. HER2 protein quantification was normalized to tumor area, and compared to receptor numbers in 12 human tumor cell lines determined by fluorescence-activated cell sorting (FACS), and with HER immunohistochemistry (IHC) test categories and histoscores in cell lines and 170 breast tumors. HER1 and HER2 expression levels determined by the VeraTag assay are proportional to receptor number over more than a 2 log10 range, and HER homodimer levels are consistent with crosslinking and immunoprecipitation results. VeraTag HER2 measurements of breast tumor tissue and cell lines correlate with standard IHC test categories (P<0.001). VeraTag HER2 levels also agree with IHC histoscores at lower HER2 protein levels, but are continuous and overlapping between IHC test categories, extending the dynamic range 5-fold to 10-fold at higher HER2 levels. The VeraTag assay specifically and reproducibly measures HER1 and HER2 protein and homodimers in FFPE tissues. The continuous measure of HER2 protein levels over a broad dynamic range, and the novel HER2 homodimer measure, are presently being assessed as predictive markers for responses to targeted HER2 therapy. PMID:19214113

Background Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. PMID:26812617

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffinembedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect

Quantifying antigens in formalin-fixed tissue is challenging and limits investigation in population-based studies of brain aging. To address this major limitation, we have developed a new technique that we call “Histelide”: immunohistochemistry (HIST-) and ELISA (-EL-) performed on a glass slide (-IDE). We validated Histelide in sections of prefrontal cortex from 20 selected cases: 12 subjects with clinically and neuropathologically diagnosed Alzheimer’s disease (AD), either autosomal dominant or late-onset forms, and 8 clinical and neuropathologic Controls. AD cases had significantly increased amyloid beta (Aβ) peptide and paired helical filament– (PHF-) tau per area of neocortex that was proteinase K-sensitive, and significantly decreased amount of synaptophysin. We next investigated prefrontal cortex from 81 consecutive cases of high cognitive performers from the Adult Changes in Thought (ACT) study, a population-based study of brain aging and incident dementia. As expected, latent AD was common in this group; however, our results quantified widely individually-varying levels of Aβ peptides and PHF-tau among these high cognitive performers. This novel approach obtains quantitative data from population-based studies, and our initial studies with high cognitive performers provide important quantitative insights into latent AD that should help guide expectations from neuroimaging and prevention studies. PMID:21999410

A gallocyanin method for demonstrating cement lines in thin, undecalcified sections of bone has been developed that is compatible with prestaining with osteochrome before plastic embedding. After sectioning at 5 microns on the Jung K heavy duty microtome, the sections are attached to a microslide using Haupt's adhesive mounting medium, placed on a slide warmer at 37 C until completely dry, and deplasticized in xylene at 45 C for 16-24 hr. Sections are stained with 0.15% gallocyanin-5% chrome alum solution for 30 min, followed by staining in buffered Villanueva blood stain for 1-1 1/2 hr, quickly dehydrated, differentiated in equal parts xylene and 100% ethanol, cleared, and mounted in Eukitt's medium. Reversal lines appear as thin, scalloped, blue or purple lines approximately 0.3 micron wide, and arrest lines as thick, homogeneous, straight or evenly curved, dark blue or purple lines approximately 2 microns wide. The method also demonstrates abnormal halo volumes around osteocytes, old and new bone matrix, osteoid seams, and the granular mineralization front at the osteoid-bone interface. It promises to be valuable in the study of age-related bone loss, osteoporosis, and metabolic bone disease. PMID:2424150

Lowicryl K4M-embedded Gram-positive and Gram-negative bacteria have a tendency to separate between the cell surface and the resin. This often leads to distortion of bacteria and more especially of mycobacteria. We describe attempts made to overcome this technical problem. Different assays were made on Bacillus subtilis, Escherichia coli, and Mycobacterium avium: 1) Modification of the bacterial surface by coating of bacteria with proteinic compounds; 2) treatment of bacteria with metallic salts known to modify cell wall polysaccharides; and 3) comparison between Lowicryl K4M and HM20. Conditions have been found in which the separation of all bacterial species from the resin is abolished. The most important factor appeared to be the treatment of bacteria before dehydration, with 0.5% uranyl acetate for 30 min. The second most important factor, especially for M. avium and to a lower extent for Gram-negative bacteria, was the use of Lowicryl HM20. No differences were observed with Gram-positive bacteria between K4M and HM20. Pre-embedding in gelatin instead of agar improved sectioning of M. avium, but had no effects on the other bacterial species. These conditions applied to macrophages infected with Shigella dysenteriae or M. avium also gave excellent results. In addition to sectioning improvement of bacteria, uranyl acetate improved the ultrastructure of bacteria and macrophages. All organelles were more clearly delineated and, hence, more easily identified. Finally, it was shown that UA treatment did not affect immunogold labeling of a variety of antigens. PMID:2110246

Rheumatoid arthritis (RA) is a chronic autoimmune disease with a heterogeneous clinical presentation affecting about 1 % of adults in developed countries. Currently, the diagnosis is based on the revised criteria of the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) from 2010. These criteria include clinical and laboratory parameters. Because of the variability of the clinical picture, delayed diagnosis of RA occurs in a significant subset of patients. Therefore, the discovery of novel biomarkers that improve the diagnosis of RA is of particular interest. Recently, it became evident that miRNAs have regulatory activities in physiologic processes and human diseases. Upregulation of miR-146a, miR-155, and miR-223 has been shown in various compartments such as serum, blood, synovial fluid, and tissues in patients with RA. A total of 87 samples were analyzed (RA 50, osteoarthritis (OA) 37). RNA was isolated from formalin-fixed paraffin-embedded synovial tissue (FFPE). The relative expression of miR-146a, miR-155, and miR-223 was determined by comparison to a housekeeping RNA molecule (snRNA U6) and an RNA pool from histologically and clinically verified OA samples. miR-146a, miR-155, and miR-223 were significantly elevated in RA compared to OA synovial tissues (p

Background Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. Methods The assay’s limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. Results A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots

The role of molecular methods in the diagnosis of head and neck cancer is rapidly evolving and holds great potential for improving outcomes for all patients who suffer from this diverse group of malignancies . However, there is considerable debate as to the best clinical approaches, particularly for Next Generation Sequencing (NGS). The choices of NGS methods such as whole exome, whole genome, whole transcriptomes (RNA-Seq) or multiple gene resequencing panels, each have strengths and weakness based on data quality, the size of the data, the turnaround time for data analysis, and clinical actionability. There have also been a variety of gene expression signatures established from microarray studies that correlate with relapse and response to treatment, but none of these methods have been implemented as standard of care for oropharyngeal squamous cell carcinoma (OPSCC). Because many genomic methodologies are still far from the capabilities of most clinical laboratories, we chose to explore the use of a combination of off the shelf targeted mutation analysis and gene expression analysis methods to complement standard anatomical pathology methods. Specifically, we have used the Ion Torrent AmpliSeq cancer panel in combination with the NanoString nCounter Human Cancer Reference Kit on 8 formalin-fixed paraffinembedded (FFPE) OPSCC tumor specimens, (4) HPV-positive and (4) HPV-negative. Differential expression analysis between HPV-positive and negative groups showed that expression of several genes was highly likely to correlate with HPV status. For example, WNT1, PDGFA and OGG1 were all over-expressed in the positive group. Our results show the utility of these methods with routine FFPE clinical specimens to identify potential therapeutic targets which could be readily applied in a clinical trial setting for clinical laboratories lacking the instrumentation or bioinformatics infrastructure to support comprehensive genomics workflows. To the best of our knowledge

Many approaches have been considered in an effort to improve the regression rate of solid fuels for hybrid rocket applications. One promising method is to use a fuel with a fast burning rate such as paraffin wax; however, additional performance increases to the fuel regression rate are necessary to make the fuel a viable candidate to replace current launch propulsion systems. The addition of energetic and/or nano-sized particles is one way to increase mass-burning rates of the solid fuels and increase the overall performance of the hybrid rocket motor.1,2 Several paraffin-based fuel grains with various energetic additives (e.g., lithium aluminum hydride (LiAlH4) have been cast in an attempt to improve regression rates. There are two major advantages to introducing LiAlH4 additive into the solid fuel matrix: 1) the increased characteristic velocity, 2) decreased dependency of Isp on oxidizer-to-fuel ratio. The testing and characterization of these solid-fuel grains have shown that continued work is necessary to eliminate unburned/unreacted fuel in downstream sections of the test apparatus.3 Changes to the fuel matrix include higher melting point wax and smaller energetic additive particles. The reduction in particle size through various methods can result in more homogeneous grain structure. The higher melting point wax can serve to reduce the melt-layer thickness, allowing the LiAlH4 particles to react closer to the burning surface, thus increasing the heat feedback rate and fuel regression rate. In addition to the formulation of LiAlH4 and paraffin wax solid-fuel grains, liquid additives of triethylaluminum and diisobutylaluminum hydride will be included in this study. Another promising fuel formulation consideration is to incorporate a small percentage of RDX as an additive to paraffin. A novel casting technique will be used by dissolving RDX in a solvent to crystallize the energetic additive. After dissolving the RDX in a solvent chosen for its compatibility

Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT-SSX1 and SYT-SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin-fixed, paraffin-embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin-embedded material. PMID:10398111

Vietnamese crude oils, including crude from the Bach Ho (White Tiger) and Dai Hung (Big Bear) fields have high paraffin content. This high-paraffin content and other characteristics of the crude oils require them to be processed through conversion units after distillation to obtain maximum light-product production. Classification of the Vietnamese crude oils as high-paraffin content used the basis of statistical analyses of fundamental properties of 400 different types of Soviet Union crude oils. The statistical analyses classified crude oils below 1 wt % paraffin content as low-paraffin crudes, between 1 wt% and 7 wt% as medium-paraffin crudes, and greater than 7 wt% as high-paraffin crudes. Evaluations of Vietnamese crude oils are ongoing. From these evaluations, the author presents some general refining properties of Vietnamese crude oils, along with the basic assays of Viet Nam's Bach Ho (White Tiger) and Dai Hung( Big Bear) crude oils.

Improved methods are described for anatomical investigation of small insects and other arthropods using serial semithin sections. The specimens were dehydrated with acidified 2,2-dimethoxypropane and embedded in ERL 4206 epoxy resin under vacuum. This procedure ensures good resin impregnation of thin, long body compartments and appendages. Furthermore, it produces excellent overall preservation of the specimen and its fragile anatomical structures. This procedure saves time and gives excellent results when sectioning difficult arthropod material. A continuous recording of serial semithin sections is possible when diamond knives are used. PMID:12713135

In order to perform correlative light and electron microscopy (CLEM) more precisely, we have modified existing specimen preparation protocols allowing fluorescence retention within embedded and sectioned tissue, facilitating direct observation across length scales. We detail a protocol which provides a precise correlation accuracy using accessible techniques in biological specimen preparation. By combining a pre-embedding uranyl acetate staining step with the progressive lowering of temperature (PLT) technique, a methacrylate embedded tissue specimen is ultrathin sectioned and mounted onto a TEM finder grid for immediate viewing in the confocal and electron microscope. In this study, the protocol is applied to rat uterine epithelial cells in vivo during early pregnancy. Correlative overlay data was used to track changes in filamentous actin that occurs in these cells from fertilization (Day 1) to implantation on Day 6 as part of the plasma membrane transformation, a process essential in the development of uterine receptivity in the rat. CLEM confirmed that the actin cytoskeleton is disrupted as apical microvilli are progressively lost toward implantation, and revealed the thick and continuous terminal web is replaced by a thinner and irregular actin band, with individually distinguishable filaments connecting actin meshworks which correspond with remaining plasma membrane protrusions. PMID:26930006

Paraffin (called kerosene in North America and other parts of the world) is the most commonly used fuel in ‎non-electrified dwellings worldwide. It is especially popular in Africa and South Asia. Although paraffin ‎offers many advantages-especially its comparatively low cost to produce-it poses two major risks of ‎injury. First, paraffin poisoning is common, either through ingestion or through inhalation of smoke and ‎fumes. Second, paraffin is highly flammable, and poses fire risk through multiple causes. This commentary ‎discusses strategies to prevent paraffin-related injury. Prevention of paraffin-related injury must be through ‎multiple strategies, and should include policy-oriented change, changes to the safety of home environments, ‎and behavioral changes targeting how individuals store and use paraffin and paraffin appliances. We review ‎successful prevention strategies in each of these domains and discuss appropriate research and community ‎initiatives that should be implemented to improve paraffin safety among at-risk populations. ‎ PMID:21483184

Microvalves containing silicone-rubber seals actuated by heating and cooling of paraffin have been proposed for development as integral components of microfluidic systems. In comparison with other microvalves actuated by various means (electrostatic, electromagnetic, piezoelectric, pneumatic, and others), the proposed valves (1) would contain simpler structures that could be fabricated at lower cost and (2) could be actuated by simpler (and thus less expensive) control systems. Each valve according to the proposal would include a flow channel bounded on one side by a flat surface and on the other side by a curved surface defined by an arched-cross-section, elastic seal made of silicone rubber [polydimethylsilane (PDMS)]. The seal would be sized and shaped so that the elasticity of the PDMS would hold the channel open except when the seal was pressed down onto the flat surface to close the channel. The principle of actuation would exploit the fact that upon melting or freezing, the volume of a typical paraffin increases or decreases, respectively, by about 15 percent. In a valve according to the proposal, the seal face opposite that of the channel would be in contact with a piston-like plug of paraffin. In the case of a valve designed to be normally open at ambient temperature, one would use a paraffin having a melting temperature above ambient. The seal would be pushed against the flat surface to close the channel by heating the paraffin above its melting temperature. In the case of a valve designed to be normally closed at ambient temperature, one would use a paraffin having a melting temperature below ambient. The seal would be allowed to spring away from the flat surface to open the channel by cooling the paraffin below its melting temperature. The availability of paraffins that have melting temperatures from 70 to +80 C should make it possible to develop a variety of normally closed and normally open valves. The figure depicts examples of prototype normally

As oil and gas production moves to deeper and colder water, subsea multiphase production systems become critical for economic feasibility. It will also become increasingly imperative to adequately identify the conditions for paraffin precipitation and predict paraffin deposition rates to optimize the design and operation of these multiphase production systems. Although several oil companies have paraffin deposition predictive capabilities for single-phase oil flow, these predictive capabilities are not suitable for the multiphase flow conditions encountered in most flowlines and wellbores. For deepwater applications in the Gulf of Mexico, it is likely that multiphase production streams consisting of crude oil, produced water and gas will be transported in a single multiphase pipeline to minimize capital cost and complexity at the mudline. Existing single-phase (crude oil) paraffin deposition predictive tools are clearly inadequate to accurately design these pipelines because they do not account for the second and third phases, namely, produced water and gas. The objective of this program is to utilize the current test facilities at The University of Tulsa, as well as member company expertise, to accomplish the following: enhance our understanding of paraffin deposition in single and two-phase (gas-oil) flows; conduct focused experiments to better understand various aspects of deposition physics; and, utilize knowledge gained from experimental modeling studies to enhance the computer programs developed in the previous JIP for predicting paraffin deposition in single and two-phase flow environments. These refined computer models will then be tested against field data from member company pipelines. The following deliverables are scheduled during the first three projects of the program: (1) Single-Phase Studies, with three different black oils, which will yield an enhanced computer code for predicting paraffin deposition in deepwater and surface pipelines. (2) Two

A technique for the examination of specimens at low electron beam intensity has been presented. Sections micrographed with this technique showed numerous knife scratches and frequently contained bands running parallel to the knife edge. Banding with an average spacing of 0.2 micro appeared to result from periodic distortion produced by impact of the knife. At the beam intensities customarily employed, differential sublimation and probably flow of the methacrylate resulted in obliteration of the bands and all but the deepest knife scratches. In addition, changes in the size, shape, and orientation of certain structures were noted. Artifacts resulting from incineration or sublimation of tissue components fixed in formalin were illustrated, and the suggestion was made that such instability to the electron beam accounted in part for the differences observed in osmium- and formalin-fixed tissues. The deformation revealed in serial sections was discussed, and it was pointed out that shortening in the axis perpendicular to the knife edge was associated with elongation in the axis parallel to the cutting edge, the elongation usually occurring locally without change in the width of the section. It was noted that the material causing contamination of the surface of sections during examination exhibited no structure but caused progressive loss of contrast. PMID:13357516

Constitutive parameter extraction from S parameter data using a rectangular waveguide whose cross section is partially filled with a material sample as opposed to being completely filled was examined. One reason for studying a partially filled geometry is to analyze the effect of air gaps between the sample and fixture for the extraction of constitutive parameters. Air gaps can occur in high temperature parameter measurements when the sample was prepared at room temperature. Single port and two port measurement approaches to parameter extraction are also discussed.

To study the distribution pattern of interleukin-6 (IL-6)-producing cells in normal human lymph nodes, we applied the in situ reverse transcription-polymerase chain reaction technique. We describe a new modification of this technique for monitoring small amounts of specific nucleotide sequences in conventional paraffinsections. This technique differs in at least two respects from those described earlier. The two decisive steps are: 1) the reverse transcription of mRNA and the subsequent amplification of cDNA by polymerase chain reaction are performed by a new single enzyme capable of both reaction types in one and the same medium without buffer exchange; and 2) for the specific detection of the amplified cDNA, a modified version of the primed in situ labeling technique was used. The technique, carried out on normal human lymph nodes, traces a low load of IL-6 mRNA in fibroblasts, endothelial cells, and a minor population of T lymphocytes in the pulp region. High levels of expression were encountered in about 20% of perisinusoidal pulp macrophages. In addition, moderate activity was detectable in sinus lining cells. Because no major activity was found in the germinal centers of the lymphoid B follicles and in the T zone, it is suggested that the plasma cell differentiation ensuing from primary and secondary B-cell immunization is mainly effected by the sinus lining cells as well as perifollicular and perisinusoidal pulp macrophages capable of producing high amounts of IL-6. Images Figure 1 Figure 2 Figure 3 PMID:9033263

As oil and gas production moves to deeper and colder water, subsea multiphase production systems become critical for economic feasibility. It will also become increasingly imperative to adequately identify the conditions for paraffin precipitation and predict paraffin deposition rates to optimize the design and operation of these multi-phase production systems. Although several oil companies have paraffin deposition predictive capabilities for single-phase oil flow, these predictive capabilities are not suitable for the multiphase flow conditions encountered in most flowlines and wellbores. For deepwater applications in the Gulf of Mexico, it is likely that multiphase production streams consisting of crude oil, produced water and gas will be transported in a single multiphase pipeline to minimize capital cost and complexity at the mudline. Existing single-phase (crude oil) paraffin deposition predictive tools are clearly inadequate to accurately design these pipelines, because they do not account for the second and third phases, namely, produced water and gas. The objective of this program is to utilize the current test facilities at The University of Tulsa, as well as member company expertise, to accomplish the following: enhance our understanding of paraffin deposition in single and two-phase (gas-oil) flows; conduct focused experiments to better understand various aspects of deposition physics; and, utilize knowledge gained from experimental modeling studies to enhance the computer programs developed in the previous JIP for predicting paraffin deposition in single and two-phase flow environments. These refined computer models will then be tested against field data from member company pipelines.

The limited success of immunogold labeling for pre-embedding immunocytochemistry of neuronal antigens is largely attributed to poor penetration of large (5–20 nm) colloidal gold particles. We examined the applicability of using silver intensification of 1 nm colloidal gold particles non-covalently bound to goat anti-rabbit immunoglobulin (1) for single labeling of a rabbit antiserum against the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), and (2) for immunogold localization of rabbit anti-TH simultaneously with immunoperoxidase labeling of a mouse monoclonal antibody against the opiate peptide, leucine-enkephalin (LE). Vibratome sections were collected from acrolein fixed brains of adult rats. These sections were immunolabeled without use of freeze-thawing or other methods that enhance penetration, but damage ultrastructure. By light microscopy, incubations in the silver intensifier (Intense M, Janssen) for less than 10 min at room temperature resulted in a brownish-red reaction product for TH. This product was virtually indistinguishable from that seen using diaminobenzidine reaction for detection of peroxidase immunoreactivity. Longer incubations produced intense black silver deposits that were more clearly distinguishable from the brown immunoperoxidase labeling. However, by light microscopy, the gold particles seen by electron microscopy were most readily distinguished from peroxidase reaction product with shorter silver intensification periods. The smaller size of gold particles with shorter periods of silver intensification also facilitated evaluation of labeling with respect to subcellular organdies. Detection of the silver product did not appear to be appreciably changed by duration of post-fixation in osmium tetroxide. In dual-labeled sections, perikarya and terminals exhibiting immunogold-silver labeling for TH were distinct from those containing immunoperoxidase labeling for LE. These results (1) define the conditions needed for

Paraffin deposition has been a problem for operators in many areas since the beginning of petroleum production from wells. An extensive literature search on paraffin problems and methods of control has been carried out, and contact was made with companies which provide chemicals to aid in the treatment of paraffin problems. A discussion of the nature of paraffins and the mechanisms of this deposition is presented. The methods of prevention and treatment of paraffin problems are summarized. Suggested procedures for handling paraffin problems are provided. Suggestions for areas of further research testing are given.

In research and clinical settings, formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue, mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features are to be related to metabolic information. Currently, high-resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI, no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one-third of the detected peaks remained unresolved by MALDI-TOF, which led to a 3-5 times lower number of m/z features compared to FTICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high-mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FTICR MSI. The systematic comparison gives rise to a synergistic combination of the different MSI platforms for high-throughput discovery and validation of biomarkers. PMID:27065343

We developed immunohistochemical and image analytical techniques to localize and quantify keratins and desmoplakins in sections of plastic-embedded human gingiva. Acetone fixation followed by plastic embedding of gingiva provided excellent morphology and permitted immunohistochemical detection of keratins 1 and 19 and desmoplakins I & II after 2.5-min trypsin digestion. Quantitative image analysis demonstrated that different volume densities of staining of each marker were associated with specific epithelial strata. Keratin 1 stained most heavily in granular strata, followed by corneal and spinous strata; keratin 19 stained most strongly in the basal layer; desmoplakins I & II stained most strongly in the granular and corneal strata. These findings confirm that variations of keratin and desmoplakin expression in these epithelial are associated with regional patterns of epithelial differentiation. PMID:1706376

The paper presents a Semi-Analytical Finite Element (SAFE) formulation coupled with a 2.5D Boundary Element Method (BEM) for the computation of the dispersion properties of viscoelastic waveguides with arbitrary cross-section and embedded in unbounded isotropic viscoelastic media. Attenuation of guided modes is described through the imaginary component of the axial wavenumber, which accounts for material damping, introduced via linear viscoelastic constitutive relations, as well as energy loss due to radiation of bulk waves in the surrounding media. Energy radiation is accounted in the SAFE model by introducing an equivalent dynamic stiffness matrix for the surrounding medium, which is derived from a regularized 2.5D boundary element formulation. The resulting dispersive wave equation is configured as a nonlinear eigenvalue problem in the complex axial wavenumber. The eigenvalue problem is reduced to a linear one inside a chosen contour in the complex plane of the axial wavenumber by using a contour integral method. Poles of leaky and evanescent modes are obtained by choosing appropriately the phase of the wavenumbers normal to the interface in compliance with the nature of the waves in the surrounding medium. Finally, the obtained eigensolutions are post-processed to compute the energy velocity and the radiated wavefield in the surrounding domain. The reliability of the method is first validated on existing results for waveguides of circular cross sectionsembedded in elastic and viscoelastic media. Next, the potential of the proposed numerical framework is shown by computing the dispersion properties for a square steel bar embedded in grout and for an H-shaped steel pile embedded in soil. PMID:23642317

... HUMAN CONSUMPTION (CONTINUED) INDIRECT FOOD ADDITIVES: ADHESIVES AND COMPONENTS OF COATINGS Substances for Use as Components of Coatings § 175.250 Paraffin (synthetic). Synthetic paraffin may be safely used as an impregnant in, coating on, or component of coatings on articles used in...

At least a portion of an oil-recovery well casing adjacent an oil-bearing earth formation is heated by the passing of an electrical current therethrough. The heated casing heats any oil entering therein. Paraffin found in the heated oil is thus maintained in a liquefied state thereby substantially reducing paraffin buildup in the oil-recovery well.

Nitrous oxide/paraffin (N2OP) hybrid rocket engines have been invented as alternatives to other rocket engines especially those that burn granular, rubbery solid fuels consisting largely of hydroxyl- terminated polybutadiene (HTPB). Originally intended for use in launching spacecraft, these engines would also be suitable for terrestrial use in rocket-assisted takeoff of small airplanes. The main novel features of these engines are (1) the use of reinforced paraffin as the fuel and (2) the use of nitrous oxide as the oxidizer. Hybrid (solid-fuel/fluid-oxidizer) rocket engines offer advantages of safety and simplicity over fluid-bipropellant (fluid-fuel/fluid-oxidizer) rocket en - gines, but the thrusts of HTPB-based hybrid rocket engines are limited by the low regression rates of the fuel grains. Paraffin used as a solid fuel has a regression rate about 4 times that of HTPB, but pure paraffin fuel grains soften when heated; hence, paraffin fuel grains can, potentially, slump during firing. In a hybrid engine of the present type, the paraffin is molded into a 3-volume-percent graphite sponge or similar carbon matrix, which supports the paraffin against slumping during firing. In addition, because the carbon matrix material burns along with the paraffin, engine performance is not appreciably degraded by use of the matrix.

Paraffin tissue microarrays (PTMAs) introduced by Kononen et al in 1998 have become a widely used technique in routine pathology and even more so in research. Kononen used a tissue puncher/arrayer (Beecher Instruments, Sun Prairie, WI, USA) to take paraffin tissue core biopsy specimens (PTCBs) of 0.6–2 mm in diameter from routine paraffin tissue blocks and transfer them to another paraffin block with up to 1000 holes. As pointed out by Mengel et al, however, it is not possible to use the Kononen/Beecher system to construct PTMAs out of archived PTCBs. To overcome this drawback in the extremely popular Beecher system, the paraffin tissue punch was modified by incorporating a conical 4 mm deep countersink. This countersink was milled with a conical precision cutter that can be bought in an ordinary hardware store (cost paraffin tissue punch and enables the construction of PTMAs with previously archived PTCBs using the widely distributed Beecher system. Moreover, this paraffin tissue punch can be used for other systems to create PTMAs, such as the low‐budget systems designed by Vogel. PMID:17079355

Celloidin mounting (embedding without infiltration) of the human central nervous system (CNS) proved to be superior to gelatin embedding for the production of serial sections ranging in thickness from 220 to 500 microm. After gallocyanin-staining, a comprehensive neuroanatomical as well as neuropathological survey of the human brain is possible, including diagnosis of Alzheimer's disease. Details of a fractionator analysis of the total striatal neuron number are described and the possible quantitative analysis of parallel immunohistochemically stained sections is discussed. PMID:11074343

A serious and common accident in rural Kenyan homesteads is accidental ingestion of paraffin when it has been mistaken for water and offered to a young child. Here we report the incidence, parental practices and outcome of severe paraffin poisoning, requiring admission at Kilifi District Hospital, Kenya. Over a 2-year period, 48 children (0.5% of all admissions) were admitted with kerosene poisoning, constituting 62% of all poisoning cases. All cases were accidental. Ten per cent had induced vomiting. One child (2%) died. We suggest these data support assessment followed by implementation of practical and affordable measures to prevent paraffin poisoning. PMID:18363584

Chemical treatments of paraffin and asphaltene deposition by means of cleaning fluids were carried out in this research project. Research focused on the characterization of asphaltene and paraffin materials and dissolution of asphaltene and paraffin deposits using surfactant/micellar fluids developed early in the project. The key parameters controlling the dissolution rate were identified and the process of asphaltene/paraffin dissolution were examined using microscopic apparatus. Numerical modeling was also carried out to understand the dissolution of paraffin deposits. The results show that fused chemical reaction systems are a promising way of removing paraffin deposits in subsea pipelines. The fused system may be in the form of alternate pulses, emulsions systems or encapsulated catalyst systems. Fused reaction systems, in fact, are extremely cost-effective--less than 10% of the cost of replacing entire sections of the blocked pipeline. The results presented in this report can have a real impact on the petroleum industry and the National Oil Program, if it is realized that the remediation technologies developed here can substantially delay abandonment (due to asphaltene/paraffin plugging) of domestic petroleum resources. The report also sheds new light on the nature and properties of asphaltenes and paraffin deposits which will ultimately help the scientific and research community to develop effective methods in eliminating asphaltene/paraffin deposition problems. It must also be realized that asphaltene remediation technologies developed and presented in this report are a real alternative to aromatic cleaning fluids currently used by the petroleum industry.

Synthetic microvascular networks are essential to enable in vitro studies of cell biology, biophysics, hemodynamics, and drug discovery, as well as in applications involving tissue engineering and artificial vasculature. But current limitations make it challenging to construct networks incorporating a hierarchy of microchannel diameters that possess cell-favored circular cross-sectional topographies. We report a new approach that overcomes these limitations by employing pressure-assisted expansion of biocompatible degradable poly(lactic acid) (PLA) substrates. This single-step process is straightforward and highly controllable, making it possible to simultaneously shape the interior topology of branched 3D and pseudo-3D microchannel networks across wide range of diameters. We further demonstrate in vitro culture of confluent endothelial cell monolayers in microchannel networks treated by this process, suggesting potential as a tool to help generate bio-mimicking vascular-like environments. PMID:24023829

Breast cancer makes up a quarter of all cancer in women, which is why research into new diagnostic methods and sample preparations need to be developed at an accelerated pace. Researchers are looking for diagnostic tools to detect when an individual has cancer cells and use that information to see what measurements and approaches can be used to take further diagnostic steps. The most common method of sample preparation is the imbibing of tumor tissue in paraffin, which can produce a background for spectroscopic measurements in the range of 500-3500 cm-1. In this study we demonstrated that proper preparation of paraffin-embedded specimens and the measurement methodology can eliminate paraffin vibration, as was done in the work Depciuch et al. 2015. Thanks to this spectroscopic technique there may become a reliable and accurate method of diagnosing breast cancer based on the evidence found from the prepared samples. The study compared the results obtained through Raman spectroscopy and FTIR (Fourier Transform Infrared) measurements of healthy and cancerous breast tissues that were either embedded in paraffin or deparaffinized. The resulting spectrum and accurate analysis led to the conclusion that the appropriate measurement of the background and the elimination of peaks from the paraffin had the greatest impact on the reliability of results. Furthermore, after the accurate, detailed studies FTIR and Raman spectroscopy on samples of breast tissue that were deparaffinized or embedded in paraffin, including a complete analysis of the peak after transformation Kramers-Kröning (KK), it was found that sample preparation did not affect the result obtained by measuring the reflectance in the mid-infrared range, and that this only had a minimal effect relating to the intensity obtained by the measurement of the Raman peak. Only in special cases, when Raman spectroscopic methods are used for research to find the peculiarities of the spectra, are deparaffinization recommended

Manganese ferrite, MnFe2O4 nanoparticles with average size of ∼6.5 nm were synthesized by using a thermal decomposition method. The nanoparticles were aggregated which was confirmed by FESEM and TEM images. The aggregates with a diameter of ∼50 nm showed interacting superspin glass (SSG) behavior. The powders were dispersed in the molten paraffin wax by using ultrasonic bath. Samples with different paraffin/ferrite weight ratios of P/F= 0, 1, 5, 10 and 20 were prepared. M-H curves of the samples revealed presence of superparamagnetic state at 300 K. Saturation magnetization (Ms) decreased from 26.6 to 1.3 emu/g by increasing the P/F value from 0 to 20, respectively. Furthermore, the VSM measurements showed a decrease in surface spin disorder of paraffin-embedded nanoparticles in comparison with bare particles. The AC magnetic susceptibility peak temperature, TP increased from 230 to >300 K with increasing the paraffin content in the samples. The present study showed that by dispersing the particles in a non-magnetic matrix, the blocking temperature could be increased.

Purpose To evaluate the postoperative outcomes of cataract eyes complicated with coexisting ocular pathologies that underwent implantation of a refractive multifocal intraocular lens (MIOL) with a surface-embedded near section. Methods LENTIS MPlus (Oculentis GmbH) refractive MIOLs were implanted in 15 eyes with ocular pathologies other than cataract (ie, six high-myopia eyes with an axial length longer than 28 mm, two fundus albipunctatus eyes, two branch retinal-vein occlusion eyes, four glaucoma eyes (one with high myopia), and two keratoconus eyes). Uncorrected or corrected distance and near visual acuity (VA) (UDVA, UNVA, CDVA, and CNVA), contrast sensitivity, and defocus curve were measured at 1 day and 6 months postoperatively, and each patient completed a 6-month postoperative questionnaire regarding vision quality and eyeglass use. Results Thirteen eyes (87%) registered 0 or better in CDVA and 12 eyes (73%) registered better than 0 in CNVA. Contrast sensitivity in the eyes of all patients was comparable to that of normal healthy subjects. No patient required eyeglasses for distance vision, but three patients (20%) required them for near vision. No patient reported poor or very poor vision quality. Conclusion With careful case selection, sectorial refractive MIOL implantation is effective for treating cataract eyes complicated with ocular pathologies. PMID:25744442

The hematoxylin and eosin (H&E) stain is the standard used for microscopic examination of tissues that have been fixed, processed, embedded, and sectioned. It can be performed manually or by automation. For economic reasons, the manual technique is generally the method of choice for facilities with a low sample volume. This protocol describes manual H&E staining of fixed, processed, paraffin-embedded, and sectioned mouse tissues. In H&E-stained tissues, the nucleic acids stain dark blue and the proteins stain red to pink or orange. For accurate phenotyping and delineation of tissue detail, the protocol must be adhered to rigorously. This includes frequent reagent changes as well as the use of "in-date" reagents. Appropriate color in a good H&E stain allows for identification of many tissue subtleties that are necessary for accurate diagnosis. PMID:24890205

Leaching tests of paraffin waste forms including boric acid, cobalt, strontium and cesium were performed to investigate the leaching characteristics of paraffin waste forms which had been generated in Korean nuclear power plants. The leaching tests were conducted according to ANSI/ANS-16.1 test procedure and the cumulative fractions leached (CFLs) of boric acid, cobalt, strontium and cesium were obtained. The compressive strength before and after the leaching test was measured for various waste forms with different mixing ratios of boric acid to paraffin. It was observed that boric acid and other nuclides immobilized within paraffin waste forms were congruently released and the leaching rates were influenced by reacted layer depth as the dissolution reaction progressed. A shrinking core model based on the diffusion-controlled dissolution kinetics was developed in order to simulate the test results. The CFLs and the leaching rates were well expressed by the shrinking core model and the cross-sectional view of specimen after the test demonstrated the applicability of this model with the shrinking dissolution front to the leaching analysis of paraffin waste forms. PMID:11300532

Background Tissue microarray technology has provided a high throughput means of evaluating potential biomarkers in archival pathological specimens. This study was carried out in order to produce tissue microarray blocks using mechanical pencil tips without high cost. Method Conventional mechanical pencil tips (Rotring Tikky II Mechanical Pencil 1.0 mm) were used to cut out 1 mm wax cylinders from the recipient block, creating from 36 to 72 holes. Three cores of tumor areas were punched out manually by using the mechanical pencil tips from donor paraffinembedded tissue blocks and transferred to the holes of the paraffin tissue microarrays. Results This technique was easy and caused little damage to the donor blocks. We successfully performed H&E slides and immunodetection without substantial tissue cylinder loss. Conclusion Our mechanical pencil tip technique is the most inexpensive easy technique among the literature. It also takes a reasonable amount of time and reduces antibody consumption during immunohistochemistry PMID:22132713

: We have developed a microwave protocol for a paraffin-embedding microtechnique of the shoot apical meristem of ZEA MAYS and have successfully applied this protocol to other plant tissues. This protocol decreases the time required for all aspects of microtechnique tissue processing, including fixation (24 hr to 15 min), dehydration (73 hr to 10 min), and infiltration (96 hr to 3 hr). Additionally, the time required to adhere paraffin ribbons to gelatin-coated slides and for the Johanson's safranin O, fast green FCF staining protocol has been significantly decreased. Using this technique, the quality of tissue preservation and subsequent in situ localization of KNOTTED mRNA was increased by using microwaves.

A lightweight armor system utilizing a face section having a multiplicity of monoliths embedded in a matrix supported on low density foam. The face section is supported with a strong stiff backing plate. The backing plate is mounted on a spall plate.

A method is disclosed for embedding auxiliary information into a set of host data, such as a photograph, television signal, facsimile transmission, or identification card. All such host data contain intrinsic noise, allowing pixels in the host data which are nearly identical and which have values differing by less than the noise value to be manipulated and replaced with auxiliary data. As the embedding method does not change the elemental values of the host data, the auxiliary data do not noticeably affect the appearance or interpretation of the host data. By a substantially reverse process, the embedded auxiliary data can be retrieved easily by an authorized user. 19 figs.

A method of embedding auxiliary information into a set of host data, such as a photograph, television signal, facsimile transmission, or identification card. All such host data contain intrinsic noise, allowing pixels in the host data which are nearly identical and which have values differing by less than the noise value to be manipulated and replaced with auxiliary data. As the embedding method does not change the elemental values of the host data, the auxiliary data do not noticeably affect the appearance or interpretation of the host data. By a substantially reverse process, the embedded auxiliary data can be retrieved easily by an authorized user.

Multiphoton microscopy (MPM) has become a powerful, important tool for tissues imaging at the molecular level. In this paper, this technique was extended to histological investigations, differentiating carcinoma in situ (CIS) lesion from normal oesophagus by imaging histological sections without hematoxylin and eosin (H&E) staining. The results show that the histology procedures of dehydration, paraffinembedding, and de-paraffinizing highlighted two photon excited fluorescence of cytoplasm and nucleolus of epithelial cell and collagen in stroma. MPM has the ability to identify the characteristics of CIS lesion including changes of squamous cells and full epithelium, identification of basement membrane, especially prominent nucleolus. The studies described here show that MPM has the potential for future retrospective studies of tumor staging by employing on histological section specimens without H&E staining.

The origin and morphological identity of hematopoietic progenitor cells, as well as their precursor, the pleuripotential hematopoietic stem cell (HSC), has not been established. Our studies of 2 microns sectioned undecalcified plastic-embedded bone marrow (BM) from healthy human fetuses; normal adults; patients with acute myeloblastic leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic granulocytic leukemia (CGL) in various stages (chronic, accelerated, acute blastic phase, and after autografting); and patients recovering from therapy-induced marrow hypoplasia suggest that proliferative hematopoietic zones exist near the endosteum (endosteal marrow) and the vascular endothelium (capillary and sinus-lining endothelium) and a maturational zone distal to these regions. In some of these areas, morphologically recognizable hematopoietic cells were seen and interpreted as emerging and maturing in a sequential progression, suggesting an origin from the endosteal or endothelial progenitors. In other loci, early hematopoietic cells were seen in close contact with the endosteal or vascular endothelial (VE) cells. This latter relationship suggested that these areas of cellular contact were important and represented sites of cell to cell interaction that may be associated with the liberation of growth factors by endosteal and endothelial cells and their action on hematopoietic progenitor cells. Following treatment-induced hypoplasia, the endosteal and VE cells were seen to modulate, transform, and migrate into the surrounding empty and edematous marrow space as fibroblasts. Later, as hemopoietic regeneration began, clusters of regenerating hematopoietic cells were seen adjacent to bone trabecule (BT) and near the vascular endothelium. We postulate that endosteal and VE cells are the equivalent of embryonal-stage, undifferentiated mesenchyme and, under the appropriate regulatory influence, are capable of modulation and transformation (differentiation) into stromal

Injection of paraffin oil to change physical configuration is an obsolete procedure from 1899, revived by bodybuilders as an alternative to intramuscular injections of steroids. Paraffin oil has destructive consequences: skin inflammation, hard oedema, sterile abscesses, diffuse lymphangitis and paraffinomas. We report a case of a 24-year-old male bodybuilder who self-injected one litre of paraffin oil in each arm. Hazard notice and advice to bodybuilders with potential risk attitude or "reverse anorexia" are warranted. PMID:20089216

Grading of cervical dysplasias at colposcopy by means of rapid frozen section avoids the delay inevitable with paraffinsections. The immediacy of the diagnosis benefits the patient, who can be treated at her first visit. A comparison of grading by frozen sections with paraffinsections has confirmed the safety of the frozen method. Additional advantages are opportunities for optimum orientation and "rescue" of specimens, improved colposcopic training, and the facilitation of special investigations on fresh cervical tissue. PMID:2863606

Extraction and high performance liquid chromatography procedures have been developed for the determination of two hindered phenols—Irganox-1010 and Santonox ®, incorporated in low density polyethylene (LDPE), paraffin oil and paraffin wax at varied concentrations. These blends were gamma irradiated from 25 to 400 kGy and the radiation and thermal stability of the antioxidant under the experimental conditions has been determined. Upon increase in radiation dose from 25 to 400 kGy, a gradual diminution in the extractable level of each antioxidant was observed. The fate of both the antioxidants in various matrices has been compared. It has been shown that both the antioxidants can influence the polyethylene network formation and the radical yield in different ways resulting in retardation in the rate of crosslinking, as determined by gel-content analysis.

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract (GIT) has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy’s solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffinembedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs compared with controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail. PMID:26708180

A method of embedding auxiliary information into the digital representation of host data created by a lossy compression technique. The method applies to data compressed with lossy algorithms based on series expansion, quantization to a finite number of symbols, and entropy coding. Lossy compression methods represent the original data as integer indices having redundancy and uncertainty in value by one unit. Indices which are adjacent in value are manipulated to encode auxiliary data. By a substantially reverse process, the embedded auxiliary data can be retrieved easily by an authorized user. Lossy compression methods use loss-less compressions known also as entropy coding, to reduce to the final size the intermediate representation as indices. The efficiency of the compression entropy coding, known also as entropy coding is increased by manipulating the indices at the intermediate stage in the manner taught by the method.

A method of embedding auxiliary information into the digital representation of host data created by a lossy compression technique is disclosed. The method applies to data compressed with lossy algorithms based on series expansion, quantization to a finite number of symbols, and entropy coding. Lossy compression methods represent the original data as integer indices having redundancy and uncertainty in value by one unit. Indices which are adjacent in value are manipulated to encode auxiliary data. By a substantially reverse process, the embedded auxiliary data can be retrieved easily by an authorized user. Lossy compression methods use loss-less compressions known also as entropy coding, to reduce to the final size the intermediate representation as indices. The efficiency of the compression entropy coding, known also as entropy coding is increased by manipulating the indices at the intermediate stage in the manner taught by the method. 11 figs.

Wax deposition on downhole equipment and in chokes, flowlines, separators, dehydration and storage equipment is a costly problem in the northern Michigan area called the Niagaran Reef trend. A number of mechanical removal techniques have been used to treat for paraffin. Among these are paraffin cutters, plunger lift, rod scrapers, hot oil or water, plastic coatings, and flowline pigging. Improvements in chemical formulation, testing, and applications have resulted in a number of economically successful chemical programs for paraffin control. Examples of field problems and solutions are presented.

A method and apparatus for embedding auxiliary information into the digital representation of host data created by a lossy compression technique and a method and apparatus for constructing auxiliary data from the correspondence between values in a digital key-pair table with integer index values existing in a representation of host data created by a lossy compression technique. The methods apply to data compressed with algorithms based on series expansion, quantization to a finite number of symbols, and entropy coding. Lossy compression methods represent the original data as ordered sequences of blocks containing integer indices having redundancy and uncertainty of value by one unit, allowing indices which are adjacent in value to be manipulated to encode auxiliary data. Also included is a method to improve the efficiency of lossy compression algorithms by embedding white noise into the integer indices. Lossy compression methods use loss-less compression to reduce to the final size the intermediate representation as indices. The efficiency of the loss-less compression, known also as entropy coding compression, is increased by manipulating the indices at the intermediate stage. Manipulation of the intermediate representation improves lossy compression performance by 1 to 10%.

A method and apparatus for embedding auxiliary information into the digital representation of host data created by a lossy compression technique and a method and apparatus for constructing auxiliary data from the correspondence between values in a digital key-pair table with integer index values existing in a representation of host data created by a lossy compression technique are disclosed. The methods apply to data compressed with algorithms based on series expansion, quantization to a finite number of symbols, and entropy coding. Lossy compression methods represent the original data as ordered sequences of blocks containing integer indices having redundancy and uncertainty of value by one unit, allowing indices which are adjacent in value to be manipulated to encode auxiliary data. Also included is a method to improve the efficiency of lossy compression algorithms by embedding white noise into the integer indices. Lossy compression methods use loss-less compression to reduce to the final size the intermediate representation as indices. The efficiency of the loss-less compression, known also as entropy coding compression, is increased by manipulating the indices at the intermediate stage. Manipulation of the intermediate representation improves lossy compression performance by 1 to 10%. 21 figs.

The identification of lymphoid surface membrane antigens in tissue sections using immunohistochemical techniques is becoming increasingly important for the diagnosis and classification of lymphoproliferative disorders. Many of the lymphocyte specific monoclonal antibodies used, however, can only be applied to frozen tissue sections. In this paper we report the successful application of a number of these antibodies to paraffin processed tissue utilizing alternative fixatives and the highly sensitive immunogold-silver staining method. The best fixatives for this purpose were formol dichromate, periodate-lysine-paraformaldehyde (PLP) and a novel fixative formed from the addition of a dichromate solution to PLP. PMID:3020216

In this work, a series of novel paraffin inhibitor, polyaminoamide (PAA), was designed and prepared by aminolysis and poly-condensation using soybean oil and canola oil as the raw material. The property of the PAAs as paraffin inhibitor was investigated, the results show several PAA samples are potent in paraffin inhibition, and PPC-2 is the most effective one. Besides, the paraffin crystal morphology analysis was carried out to provide the mechanism of paraffin inhibition. PMID:22152091

Breast cancer is the most frequently occurring disease among women worldwide. The early stage of breast cancer identification is the key challenge in cancer control and prevention procedures. Although gene expression profiling helps to understand the molecular mechanism of diseases or disorder in the living system, gene expression pattern alone is not sufficient to predict the exact mechanisms. Current proteomics tools hold great application for analysis of cancerous conditions. Hence, the generation of differential protein expression profiles has been optimized for breast cancer and normal tissue samples in our organization. Normal and tumor tissues were collected from 20 people from a local hospital. Proteins from the diseased and normal tissues have been investigated by 2D gel electrophoresis and MALDI-TOF-MS. The peptide mass fingerprint data were fed into various public domains like Mascot, MS-Fit, and Pept-ident against Swiss-Prot protein database and the proteins of interest were identified. Some of the differentially expressed proteins identified were human annexin, glutathione S-transferase, vimentin, enolase-1, dihydrolipoamide dehydrogenase, glutamate dehydrogenase, Cyclin A1, hormone sensitive lipase, beta catenin, and so forth. Many types of proteins were identified as fundamental steps for developing molecular markers for diagnosis of human breast cancer as well as making a new proteomic database for future research. PMID:27110560

Nine cutaneous solitary neurofibromas have been studied using antibodies against vimentin, S 100 protein, lysozyme, myoglobin, factor VIII, neurofilament, neuron specific enolase, and myelin-associated antigen. Most of the tumor cells showed positive reactions to S 100 protein and vimentin with different patterns of staining. Whereas vimentin was detected in the cell periphery, S 100 protein was concentrated in the perinuclear area and distinct in the cytoplasm. About 60 percent of the tumor cells revealed positive staining for laminin. Myoglobin, neurofilament, and neuron specific enolase could not be proved in the tumor tissue. Our results suggest that the majority of neurofibroma cells may derive from Schwann's cells. PMID:3529668

Breast cancer is the most frequently occurring disease among women worldwide. The early stage of breast cancer identification is the key challenge in cancer control and prevention procedures. Although gene expression profiling helps to understand the molecular mechanism of diseases or disorder in the living system, gene expression pattern alone is not sufficient to predict the exact mechanisms. Current proteomics tools hold great application for analysis of cancerous conditions. Hence, the generation of differential protein expression profiles has been optimized for breast cancer and normal tissue samples in our organization. Normal and tumor tissues were collected from 20 people from a local hospital. Proteins from the diseased and normal tissues have been investigated by 2D gel electrophoresis and MALDI-TOF-MS. The peptide mass fingerprint data were fed into various public domains like Mascot, MS-Fit, and Pept-ident against Swiss-Prot protein database and the proteins of interest were identified. Some of the differentially expressed proteins identified were human annexin, glutathione S-transferase, vimentin, enolase-1, dihydrolipoamide dehydrogenase, glutamate dehydrogenase, Cyclin A1, hormone sensitive lipase, beta catenin, and so forth. Many types of proteins were identified as fundamental steps for developing molecular markers for diagnosis of human breast cancer as well as making a new proteomic database for future research. PMID:27110560

To study the relationship between emulsion stability and polymer emulsifier concentration, the preparation of paraffin oil emulsions by hydroxypropyl methylcellulose (HPMC) was carried out with HPMC concentrations below the overlapping concentration (C(*)) of HPMC. The stability of the emulsions incorporating HPMC was investigated by measuring the creaming velocity, volume fraction of emulsified paraffin oil, oil droplet size, and some rheological responses such as the stress-strain sweep curve and strain and frequency dependences of dynamic viscoelastic moduli. The paraffin oil was almost emulsified by HPMC above C(*)/20: the volume fraction of paraffin oil in the emulsion was higher than 0.72. Increasing in the HPMC concentration led to decreases in both the average oil droplet size and creaming velocity and an increase in the yield stress. All emulsions behaved as solid-like viscoelastic matter. Additionally, the measured dynamic storage moduli were compared with those calculated from a relationship based on functions of the volume fraction of oil in the emulsions and Laplace pressure; good agreement between the measured and calculated moduli was obtained. On the other hand, at HPMC concentrations below C(*)/50, the emulsified paraffin oil became unstable and the oil and the HPMC solution eventually separated. PMID:22138268

Parallel percutaneous absorption studies of two 14C-labelled chlorinated paraffins (C18, 50-53% chlorination; C28, 47% chlorination) were carried out in the Sprague-Dawley rat. The dermally applied dose (66 mg/cm2) was approximately equivalent to 2.0 g/kg of body weight. An oral absorption study with the C18-chlorinated paraffin (0.5 g/kg) was carried out in rats for comparison. Less than 1% of the dermally applied dose of [1-14C]polychlorooctadecane (50-53% chlorination) and less than 0.1% of the applied dose of [14,15-14C]polychlorooctacosane (47% chlorination) were recovered in excreta, expired air and tissues after 96 hours. In contrast, approximately 86% of the orally administered dose of [1-14C]polychlorooctadecane (0.5 g/kg) was recovered. These results indicate that rat skin acts as an effective barrier to chlorinated paraffins containing eighteen or more carbons and more than 40% chlorine by weight. The oral absorption of the C18 chlorinated paraffin can be estimated to be nearly 100 times greater than its dermal absorption. Based on current toxicity results from rodent experiments and these present findings, chlorinated paraffins of the type tested would be expected to have little or no effect in animals as a result of dermal exposure. It is reasonable to assume that such chlorinated paraffins are unlikely to be systemically toxic to humans by skin contact under normal conditions of production and use. PMID:3686542

MCCPs (C14 – C17) and the C18-20 LCCPs are liquid mixtures of chlorinated alkanes. Short chain chlorinated paraffins (SCCPs, C10-C13) have been the focus of coordinated global action (including by US EPA as an action plan chemical), and MCCPs and LCCPs are alternatives to SCCPs f...

Enrichment and monitoring of the above-described cultures for the ability to degrade waxy paraffins under anaerobic conditions will continue. Experiments will be conducted to determine the nutritional requirements of some of the enrichment cultures to improve growth and deduce...

A series of commercial extreme pressure (EP) additives containing sulfur, phosphorus, and chlorine were investigated for their EP properties in soybean (SBO) and paraffinic (PRFN) base oils. The investigations were conducted using a 4-ball (4B) and twist-compression (TC) tribometers. The concentra...

On-tissue digestion matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can be used to record spatially correlated molecular information from formalin-fixed, paraffin-embedded (FFPE) tissue sections. In this work, we present the in situ multimodal analysis of N-linked glycans and proteins from the same FFPE tissue section. The robustness and applicability of the method are demonstrated for several tumors, including epithelial and mesenchymal tumor types. Major analytical aspects, such as lateral diffusion of the analyte molecules and differences in measurement sensitivity due to the additional sample preparation methods, have been investigated for both N-glycans and proteolytic peptides. By combining the MSI approach with extract analysis, we were also able to assess which mass spectral peaks generated by MALDI-MSI could be assigned to unique N-glycan and peptide identities. PMID:27373711

We describe a method of biotin/avidin-peroxidase detection using second and third stage reagents in Coplin jars. This method allows a large quantity of sections to be stained simultaneously with a minimal amount of technical time involved. A wide range of mouse monoclonal antibodies of varying specificities and isotypes were used to stain both frozen and paraffin-embeddedsections of various normal and neoplastic tissues. Three different biotinylated anti-mouse antibodies were tested, including F(ab')2 antibody fragments of one, followed by horseradish peroxidase conjugated avidin. All monoclonal antibodies employed gave good staining, using incubation times of 30-50 minutes. The staining was done during a mean period of 25 to 27 days with an average staining load of 500 sections per Coplin jar. PMID:2420169

Paraffin deposition problems, that have plagued the oil industry, are currently remediated by mechanical and chemical means. However, since these methods are problematic, a microbiological approach has been considered. The bacteria, required for the mitigation of paraffin deposition problems, should be able to survive the high temperatures of oil wells and degrade the paraffins under low oxygen and nutrient conditions while sparing the low carbon chain paraffins. In this study, a thermophilic paraffinic wax degrading bacterial strain was isolated from a soil sample contaminated with paraffinic crude oil. The selected strain, Geobacillus TERI NSM, could degrade 600mg of paraffinic wax as the sole carbon source in 1000ml minimal salts medium in 7d at 55 degrees C. This strain was identified as Geobacillus kaustophilus by fatty acid methyl esters analysis and 16S rRNA full gene sequencing. G. kaustophilus TERI NSM showed 97% degradation of eicosane, 85% degradation of pentacosane and 77% degradation of triacontane in 10d when used as the carbon source. The strain TERI NSM could also degrade the paraffins of crude oil collected from oil wells that had a history of paraffin deposition problems. PMID:17942139

Purpose: The purpose of this study was to explore the effects of a paraffin screen located at various positions in the maze on the neutron dose equivalent at the maze door.Methods: The neutron dose equivalent was measured at the maze door of a room containing a 15 MV linear accelerator for x-ray therapy. Measurements were performed for several positions of the paraffin screen covering only 27.5% of the cross-sectional area of the maze. The neutron dose equivalent was also measured at all screen positions. Two simple models of the neutron source were considered in which the first assumed that the source was the cross-sectional area at the inner entrance of the maze, radiating neutrons in an isotropic manner. In the second model the reduction in the neutron dose equivalent at the maze door due to the paraffin screen was considered to be a function of the mean values of the neutron fluence and energy at the screen.Results: The results of this study indicate that the equivalent dose at the maze door was reduced by a factor of 3 through the use of a paraffin screen that was placed inside the maze. It was also determined that the contributions to the dosage from areas that were not covered by the paraffin screen as viewed from the dosimeter, were 2.5 times higher than the contributions from the covered areas. This study also concluded that the contributions of the maze walls, ceiling, and floor to the total neutron dose equivalent were an order of magnitude lower than those from the surface at the far end of the maze.Conclusions: This study demonstrated that a paraffin screen could be used to reduce the neutron dose equivalent at the maze door by a factor of 3. This paper also found that the reduction of the neutron dose equivalent was a linear function of the area covered by the maze screen and that the decrease in the dose at the maze door could be modeled as an exponential function of the product φ·E at the screen.

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffinembedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants. PMID:26505195

Fast-scan cyclic voltammetry (FSCV) is a powerful technique for measuring sub-second changes in neurotransmitter levels. A great time-limiting factor in the use of FSCV is the production of high-quality recording electrodes; common recording electrodes consist of cylindrical carbon fiber encased in borosilicate glass. When the borosilicate is heated and pulled, the molten glass ideally forms a tight seal around the carbon fiber cylinder. It is often difficult, however, to guarantee a perfect seal between the glass and carbon. Indeed, much of the time spent creating electrodes is in an effort to find a good seal. Even though epoxy resins can be useful in this regard, they are irreversible (seals are permanent), wasteful (epoxy cannot be reused once hardener is added), hazardous (hardeners are often caustic), and require curing. Herein we characterize paraffin as an electrode sealant for FSCV microelectrodes. Paraffin boasts the advantages of near-immediate curing times, simplicity in use, long shelf-life and stable waterproof seals capable of withstanding extended cycling. Borosilicate electrode tips were left intact or broken and dipped in paraffinembedding wax. Excess wax was removed from the carbon surface with xyelenes or by repeated cycling at an extended waveform (-0.4 to 1.4V, 400 V/s, 60 Hz). Then, the waveform was switched to a standard waveform (-0.4 to 1.3V, 400 V/s, 10 Hz) and cycled until stable. Wax-sealing does not inhibit electrode sensitivity, as electrodes detected linear changes in dopamine before and after wax (then xylenes) exposure. Paraffin seals are intact after 11 days of implantation in the mouse, and still capable of measuring transient changes in in vivo dopamine. From this it is clear that paraffin wax is an effective sealant for FSCV electrodes that provides a convenient substitute to epoxy sealants. PMID:26505195

The paper presents description and analysis of the results obtained in the investigation performed on a disc-ball tribotester T-11. Samples of 100Cr6 steel were tested, while as lubricant the mixtures of paraffin oil, with addition of 0.5%, 1%, and 2% of liquid crystalline compounds, from two homologous series defined with nOBCAB and nCBB symbols, were used. The friction force and wear of a sample and a counter-sample were measured. The improvement in tribological and anti-wear properties was found for all mixtures in relation to paraffin oil. The best tribological properties and the best wearability were obtained for mixtures with a compound 8CBB. This compound differs from the others in formation of different liquid crystalline phases.

A facile method that allows for Ni(cod)2 to be used on the benchtop is reported. The procedure involves the preparation of paraffin-Ni(cod)2 capsules, which are stable to air and moisture. It is demonstrated that these readily available capsules can be used to promote a range of Ni(cod)2-catalyzed transformations. These studies are expected to promote the further use of Ni(cod)2 in organic synthesis. PMID:27454146

Neuron counting in the cochlea is a crucial but time-consuming operation for which various methods have been developed. To improve simplicity and efficiency, we tested an imaging method of the cochlea, and based on Confocal Laser Scanning Microscopy (CLSM), we visualised Rosenthal's Canal and quantified the spiral ganglion neurons (SGN) within. Cochleae of 8 normal hearing guinea pigs and one implanted with a silicone filament were fixed in paraformaldehyde (PFA), decalcified, dehydrated and cleared in Spalteholz solution. Using the tissue's autofluorescence, CLSM was performed at 100 fold magnification generating z-series stacks of about 20 slices of the modiolus. In 5 midmodiolar slices per cochlea the perimeters of the Rosenthal's Canal were surveyed, representative neuron diameters were measured and the neurons first counted manually and then software-assisted. For comparison, 8 normal hearing guinea pig cochleae were embedded in paraffin and examined similarly. The CLSM method has the advantage that the cochleae remain intact as an organ and keep their geometrical structure. Z-stack creation is nearly fully-automatic and frequently repeatable with various objectives and step sizes and without visible bleaching. The tissue shows minimal or no shrinking artefacts and damage typical of embedding and sectioning. As a result, the cells in the cleared cochleae reach an average diameter of 21 μm and a density of about 18 cells/10,000 μm(2) with no significant difference between the manual and the automatical counts. Subsequently we compared the CLSM data with those generated using the established method of paraffin slides, where the SGN reached a mean density of 9.5 cells/10,000 μm(2) and a mean soma diameter of 13.6 μm. We were able to prove that the semi-automatic CLSM method is a simple and effective technique for auditory neuron count. It provides a high grade of tissue preservation and the automatic stack-generation as well as the counter software reduces

This work reports on the process optimization of ultrasound-assisted, paraffin wax in water nanoemulsions, stabilized by modified sodium dodecyl sulfate (SDS). This work focuses on the optimization of major emulsification process variables including sonication time, applied power and surfactant concentration. The effects of these variables were investigated on the basis of mean droplet diameter and stability of the prepared emulsion. It was found that the stable emulsion with droplet diameters about 160.9 nm could be formed with the surfactant concentration of 10 mg/ml and treated at 40% of applied power (power density: 0.61 W/ml) for 15 min. Scanning electron microscopy (SEM) was used to study the morphology of the emulsion droplets. The droplets were solid at room temperature, showing bright spots under polarized light and a spherical shape under SEM. The electrophoretic properties of emulsion droplets showed a negative zeta potential due to the adsorption of head sulfate groups of the SDS surfactant. For the sake of comparison, paraffin wax emulsion was prepared via emulsion inversion point method and was checked its intrinsic stability. Visually, it was found that the emulsion get separated/creamed within 30 min. while the emulsion prepared via ultrasonically is stable for more than 3 months. From this study, it was found that the ultrasound-assisted emulsification process could be successfully used for the preparation of stable paraffin wax nanoemulsions. PMID:25465097

Real-time three-dimensional (3D) reconstruction of epithelial structures in human mammary gland tissue blocks mapped with selected markers would be an extremely helpful tool for breast cancer diagnosis and treatment planning. Besides its clear clinical application, this tool could also shed a great deal of light on the molecular basis of breast cancer initiation and progression. In this paper we present a framework for real-time segmentation of epithelial structures in two-dimensional (2D) images of sections of normal and neoplastic mammary gland tissue blocks. Complete 3D rendering of the tissue can then be done by surface rendering of the structures detected in consecutive sections of the blocks. Paraffinembedded or frozen tissue blocks are first sliced, and sections are stained with Hematoxylin and Eosin. The sections are then imaged using conventional bright field microscopy and their background is corrected using a phantom image. We then use the Fast-Marching algorithm to roughly extract the contours of the different morphological structures in the images. The result is then refined with the Level-Set method which converges to an accurate (sub-pixel) solution for the segmentation problem. Finally, our system stacks together the 2D results obtained in order to reconstruct a 3D representation of the entire tissue block under study. Our method is illustrated with results from the segmentation of human and mouse mammary gland tissue samples.

Abstract Objective To explore what individuals at risk of injury from using paraffin (also known as kerosene) know about paraffin safety, what they do to protect themselves and their families from paraffin-related injury, and how they perceive their risk for such injury. Also, to explore interrelations between these factors and age, sex, education and income. Methods A sample of 238 individuals was randomly recruited from low-income housing districts near Cape Town, South Africa in 2007. Trained research assistants interviewed participants to explore their knowledge about paraffin-related safety and their perceived risk of injury from using paraffin. Researchers inspected participants’ homes to evaluate paraffin safety practices. Descriptive and correlational analyses were conducted. Findings Participants had relatively low levels of knowledge about paraffin-related safety. They had high levels of unsafe practice and their perceived risk of injury was moderate. Knowledge of paraffin safety and safe practices were positively correlated with each other. Greater knowledge showed a negative correlation with the perception of being at risk for injury, but safe practices showed no correlation with perceived risk of injury. Formal education, the number of children in the home and frequency of paraffin use were positively correlated with knowledge but not with safe practices. The only significant correlate to safe practices was greater income, perhaps a reflection of the impact of financial resources on paraffin safety practices. Conclusion To develop successful paraffin safety interventions, it is necessary to understand baseline levels of knowledge, practice and perceived risk of injury among at-risk populations. Our findings could be of value for designing interventions that will increase knowledge, improve safe practices and lead to the accurate perception of the risk of injury from using paraffin. PMID:19784450

Two bacterial strains, paraffin removal strain and biosurfactant-producing strain, named BHJ-1 and QFL-1, were isolated from oil production wells in Daqing oilfield of China. They were subsequently identified as Bacillus cereus QAU68 and Bacillus subtilis XCCX, respectively. As an indicator of the degradation paraffin, the inoculum concentration of BHJ-1 and QFL-1 were added in different proportions, the optimum proportion was 5:2. In this proportion the degradation rate of paraffin could reach 64 %, the prevention rate of paraffin could reach 55 %. PMID:24426154

By scanning biological tissues in vivo and in vitro with optical coherence tomography, it is found that liquid paraffin can enhance the percutaneous penetration of glycerol in deep layers of tissue and take synergistically optical clearing effect with glycerol. It is shown from experimental results that 30% - 50% liquid paraffin glycerol solutions have the best enhancement effect. Considering the refractive index of liquid paraffin and its medicinal value, we think liquid paraffin will play an important role in optical clearing as the penetration enhancer of glycerol in future clinical research. PMID:21833369

Data embedding is a new steganographic method for combining digital information sets. This paper describes the data embedding method and gives examples of its application using software written in the C-programming language. Sandford and Handel produced a computer program (BMPEMBED, Ver. 1.51 written for IBM PC/AT or compatible, MS/DOS Ver. 3.3 or later) that implements data embedding in an application for digital imagery. Information is embedded into, and extracted from, Truecolor or color-pallet images in Microsoft{reg_sign} bitmap (.BMP) format. Hiding data in the noise component of a host, by means of an algorithm that modifies or replaces the noise bits, is termed {open_quote}steganography.{close_quote} Data embedding differs markedly from conventional steganography, because it uses the noise component of the host to insert information with few or no modifications to the host data values or their statistical properties. Consequently, the entropy of the host data is affected little by using data embedding to add information. The data embedding method applies to host data compressed with transform, or {open_quote}lossy{close_quote} compression algorithms, as for example ones based on discrete cosine transform and wavelet functions. Analysis of the host noise generates a key required for embedding and extracting the auxiliary data from the combined data. The key is stored easily in the combined data. Images without the key cannot be processed to extract the embedded information. To provide security for the embedded data, one can remove the key from the combined data and manage it separately. The image key can be encrypted and stored in the combined data or transmitted separately as a ciphertext much smaller in size than the embedded data. The key size is typically ten to one-hundred bytes, and it is in data an analysis algorithm.

Emerging studies demonstrate that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. DNA fluorescent in situ hybridization (FISH) is a common molecular technique used to visualize the location of DNA sequences. The vast majority of DNA FISH studies are conducted on cultured cells due to the technical difficulties encountered using fixed tissue sections. However, the use of cultured cells poses important limitations that could yield misleading results, making in vivo analysis a far superior approach. Here we present a protocol for multiplexed three dimensional DNA FISH in mouse brain sections, which is also applicable to other tissues. Paraffin-embedded tissues could be used but the embedding and preparation of the samples is time-consuming and often associated with poor antigenicity. To overcome this problem we:•developed a FISH technique using fixed, frozen cryosections;•provide specific instructions for tissue processing for proper fixation and freezing, including equilibration in sucrose gradients to maintain proper cellular structure;•include optimized permeabilization and washing steps to achieve specific signal and to limit background fluorescence in tissue sections. PMID:26150931

Formalin-fixed and paraffin-embedded (FFPE) tissues present a particular challenge for proteomic analysis. Yet, most of the archived tissues in hospitals and tissue banks worldwide are only available in this form. We have developed conditions for removal of the embedding medium and protein digestion, such that informative tryptic peptides are released from fixed proteins which are suitable for analysis by liquid chromatography-mass spectrometry (LC-MS). We demonstrate that the peptide identifications made by this approach compare favorably to those made from matched fresh frozen tissue. Moreover, we demonstrate that a high level of sequence coverage can be observed for proteins of interest. PMID:16335994

High performance active materials are of rapidly growing interest for applications including soft robotics, microfluidic systems, and morphing composites. In particular, paraffin wax has been used to actuate miniature pumps, solenoid valves, and composite fibers, yet its deployment is typically limited by the need for external volume constraint. We demonstrate that compact, high-performance paraffin actuators can be made by confining paraffin within vertically aligned carbon nanotube (CNT) films. This large-stroke vertical actuation is enabled by strong capillary interaction between paraffin and CNTs and by engineering the CNT morphology by mechanical compression before capillary-driven infiltration of the molten paraffin. The maximum actuation strain of the corrugated CNT-paraffin films (∼0.02-0.2) is comparable to natural muscle, yet the maximum stress is limited to ∼10 kPa by collapse of the CNT network. We also show how a CNT-paraffin film can serve as a self-activating thermal interface that closes a gap when it is heated. These new CNT-paraffin film actuators could be produced by large-area CNT growth, infiltration, and lamination methods, and are attractive for use in miniature systems due to their self-contained design. PMID:25822633

A method to produce silica embedded metal hydride was developed. The product is a composite in which metal hydride particles are embedded in a matrix of silica. The silica matrix is highly porous. Hydrogen gas can easily reach the embedded metal hydride particles. The pores are small so that the metal hydride particles cannot leave the matrix. The porous matrix also protects the metal hydride particles from larger and reactive molecules such as oxygen, since the larger gas molecules cannot pass through the small pores easily. Tests show that granules of this composite can absorb hydrogen readily and withstand many cycles without making fines.

Additives, called also intentional impurities, act on crystal growth kinetics. Generally, they are very specific and some act as inhibitors, others as promoters. Paraffin solution growth is sensitive to organic additive molecules and especially to polymers. In the latter case, the same molecule is able to act both as an inhibitor and as a promoter, depending only on its degree of polymerisation m. Polyalkylacrylate with low m value slows down the precipitation kinetics of n-paraffins, with species forming mestable solid solutions. On the contrary, high m values promote the precipitation, provided the polymer crystallizes prior to paraffin precipitation. In this case the polymer crystals act as epitaxial seeds, leading to paraffin crystal agglomerates of large size. This versatility of some polymers is of great help in industrial processes where paraffin crystals are involved.

Paraffin is a common deposit in oil production pipeline. It occurs when the oil flowing-temperature is under Wax Appearance Temperature (WAT) or pour-point temperature. Several prediction models so far only estimatethe location where the paraffin-wax is possibly formed and there is no prediction about paraffin-wax growth over time. Therefore, this paper presents a new mathematical model to accurately predict paraffin-wax growth in oil production pipeline. The proposed model contains stochastic and kriging method. The stochastic model is developed based on Markov and Poisson model and used to describe the generation time and growth of scale. Kriging model is then combined to describe the position of scale along the production pipeline. As the result of the combined model, paraffin-wax thickness can be mapped in space and time. This prediction is important to determine and decide an effective production operation and efficient investment.

Hyperelastic sensors are fabricated by embedding a silicone rubber film with microchannels of conductive liquid. In the case of soft tactile sensors, pressing the surface of the elastomer will deform the cross-section of underlying channels and change their electrical resistance. Soft pressure sensors may be employed in a variety of applications. For example, a network of pressure sensors can serve as artificial skin by yielding detailed information about contact pressures. This concept was demonstrated in a hyperelastic keypad, where perpendicular conductive channels form a quasi-planar network within an elastomeric matrix that registers the location, intensity and duration of applied pressure. In a second demonstration, soft curvature sensors were used for joint angle proprioception. Because the sensors are soft and stretchable, they conform to the host without interfering with the natural mechanics of motion. This marked the first use of liquid-embedded elastomer electronics to monitor human or robotic motion. Finally, liquid-embedded elastomers may be implemented as conductors in applications that call for flexible or stretchable circuitry, such as robotic origami.

We have realized and studied a rubidium atomic frequency standard based on a paraffin-coated cell, exhibiting a short-term frequency stability <3 Multiplication-Sign 10{sup -12} {tau}{sup -1/2} between {tau} = 1 and 100 s. Characterization of the wall-coating is performed by measuring the T{sub 1} and T{sub 2} relaxation times. Perturbations of the medium- to long-term clock stability, due to variations in the laser-intensity, laser frequency, the microwave power shift, and the shifts due to temperature variations are measured and analyzed. A method for reducing the intensity light-shift by detuning the laser frequency and the resulting improvement in clock stability is demonstrated. This work is of relevance for further improvements on Rb cell standards using anti-relaxation wall-coating technology.

The invention is directed to an ionic liquid comprising (i) a cationic portion containing a complex of a silver (I) ion and one or more neutral ligands selected from organoamides, organoamines, olefins, and organonitriles, and (ii) an anionic portion having the chemical formula ##STR00001## wherein m and n are independently 0 or an integer of 1 or above, and p is 0 or 1, provided that when p is 0, the group --N--SO.sub.2--(CF.sub.2).sub.nCF.sub.3 subtended by p is replaced with an oxide atom connected to the shown sulfur atom. The invention is also directed to a method for separating an olefin from an olefin-paraffin mixture by passing the mixture through a layer of the ionic liquid described above.

The invention is directed to an ionic liquid comprising (i) a cationic portion containing a complex of a silver (I) ion and one or more neutral ligands selected from organoamides, organoamines, olefins, and organonitriles, and (ii) an anionic portion having the chemical formula ##STR00001## wherein m and n are independently 0 or an integer of 1 or above, and p is 0 or 1, provided that when p is 0, the group --N--SO.sub.2--(CF.sub.2).sub.nCF.sub.3 subtended by p is replaced with an oxide atom connected to the shown sulfur atom. The invention is also directed to a method for separating an olefin from an olefin-paraffin mixture by passing the mixture through a layer of the ionic liquid described above.

Antirelaxation coatings in atomic vapor cells allow ground-state coherent spin states to survive many collisions with the cell walls. This reduction in the ground-state decoherence rate gives rise to ultranarrow-bandwidth features in electromagnetically induced transparency (EIT) spectra, which can form the basis of, for example, long-time scale slow and stored light, sensitive magnetometers, and precise frequency standards. Here we study, both experimentally and theoretically, how Zeeman EIT contrast and width in paraffin-coated rubidium vapor cells are determined by cell and laser-beam geometry, laser intensity, and atomic density. Using a picture of Ramsey pulse sequences, where atoms alternately spend ''bright'' and ''dark'' time intervals inside and outside the laser beam, we explain the behavior of EIT features in coated cells, highlighting their unique characteristics and potential applications.

Using time-resolved small-angle neutron scattering (SANS), the time-dependent microphase separation occurring in metastable, quenched binary paraffin mixtures C{sub 30}H(D){sub 62}/C{sub 36}D(H){sub 74} doped into porous graphite has been observed. In the presence of graphite, microphase formation is enhanced compared to the bulk mixtures and the isotopic dependence of the demixing process reported for these systems when quenched to 20{degree}C is not apparent. We relate the enhanced microphase separation to an elevation of the eutectic temperature relative to the critical temperature, due to stabilization of the paraffins at the graphite basal plane. For 1:1 mixtures, the microphase forms an alternating lamellar structure, while the 1:4 and 4:1 mixtures exhibit an increase in scattering at lower angles associated with significantly longer repeat-spacings. An increase in quench temperature from 20 to 27{degree}C increases the strength of the microphase scattering over the time period studied, but quenching to 35{degree}C results in a significant reduction in this signal. On aging, additional weaker peaks are observed, which for 1:1 mixtures, are consistent with the formation of alternating lamellae. For all mixtures, except 1:4 C{sub 30}H{sub 62}/C{sub 36}D{sub 74}, there is a constant offset in Q between the strong and weak peaks. The scattering can be understood to rise from a mixed lamellar system in which incommensurate deviations from the mean structure occur. 56 refs., 14 figs., 9 tabs.

In this paper we present a scheme for real time segmentation of histological structures in microscopic images of normal and neoplastic mammary gland sections. Paraffinembedded or frozen tissue blocks are sliced, and sections are stained with hematoxylin and eosin (H&E). The sections are then imaged using conventional bright field microscopy. The background of the images is corrected by arithmetic manipulation using a ''phantom.'' Then we use the fast marching method with a speed function that depends on the brightness gradient of the image to obtain a preliminary approximation to the boundaries of the structures of interest within a region of interest (ROI) of the entire section manually selected by the user. We use the result of the fast marching method as the initial condition for the level set motion equation. We run this last method for a few steps and obtain the final result of the segmentation. These results can be connected from section to section to build a three-dimensional reconstruction of the entire tissue block that we are studying.

In this paper we present a scheme for real time segmentation of histological structures in microscopic images of normal and neoplastic mammary gland sections. Paraffinembedded or frozen tissue blocks are sliced, and sections are stained with hematoxylin and eosin (H&E). The sections are then imaged using conventional bright field microscopy. The background of the images is corrected by arithmetic manipulation using a "phantom." Then we use the fast marching method with a speed function that depends on the brightness gradient of the image to obtain a preliminary approximation to the boundaries of the structures of interest within a region of interest (ROI) of the entire section manually selected by the user. We use the result of the fast marching method as the initial condition for the level set motion equation. We run this last method for a few steps and obtain the final result of the segmentation. These results can be connected from section to section to build a three-dimensional reconstruction of the entire tissue block that we are studying.

Paraffin wax and aqueous paraffin emulsions can be used as controlled release carriers for insect sex pheromones for mating disruption of orchard pests. Paraffin can be applied at ambient temperature as an aqueous emulsion, adheres to tree bark or foliage, releases pheromone for an extended period of time, and will slowly erode from bark and biodegrade in soil. Pheromone emulsions can be applied with simple spray equipment. Pheromone release-rates from paraffin were measured in laboratory flow-cell experiments. Pheromone was trapped from an air stream with an adsorbent, eluted periodically, and quantified by gas chromatography. Pheromone release from paraffin was partition-controlled, providing a constant (zero-order) release rate. A typical paraffin emulsion consisted of 30% paraffin, 4% pheromone, 4% soy oil, 1% vitamin E, 2% emulsifier, and the balance water. Soy oil and vitamin E acted as volatility suppressants. A constant release of oriental fruit moth pheromone from paraffin emulsions was observed in the laboratory for more than 100 days at 27 degreesC, with release-rates ranging from 0.4 to 2 mg/day, depending on the concentration and surface area of the dried emulsion. The use of paraffin emulsions is a viable method for direct application of insect pheromones for mating disruption. Sprayable formulations can be designed to release insect pheromones to the environment at a rate necessary for insect control by mating disruption. At temperatures below 38 degreesC, zero-order release was observed. At 38 degreesC and higher, pheromone oxidation occurred. A partition-controlled release mechanism was supported by a zero-order pheromone release-rate, low air/wax partition coefficients, and pheromone solubility in paraffin. PMID:9895411

Quantitative analysis of electron microscope images of organic and biological two-dimensional crystals has previously shown that the absolute contrast reached only a fraction of that expected theoretically from the electron diffraction amplitudes. The accepted explanation for this is that irradiation of the specimen causes beam-induced charging or movement, which in turn causes blurring of the image due to image or specimen movement. In this paper, we used three different approaches to try to overcome this image-blurring problem for monolayer crystals of paraffin. Our first approach was to use an extreme form of spotscan imaging, in which a single image was assembled on film by the successive illumination of up to 50,000 spots each of diameter around 7nm. The second approach was to use the Medipix II detector with its zero-noise readout to assemble a time-sliced series of images of the same area in which each frame from a movie with up to 400 frames had an exposure of only 500 electrons. In the third approach, we simply used a much thicker carbon support film to increase the physical strength and conductivity of the support. Surprisingly, the first two methods involving dose fractionation respectively in space or time produced only partial improvements in contrast whereas the third approach produced many virtually perfect images, in which the absolute contrast predicted from the electron diffraction amplitudes was observed in the images. We conclude that it is possible to obtain consistently almost perfect images of beam-sensitive specimens if they are attached to an appropriately strong and conductive support, but great care is needed in practice and the problem of how best to image ice-embedded biological structures in the absence of a strong, conductive support film requires more work. PMID:21185452

Autofluorescence exhibited by tissues often interferes with immunofluorescence. Using imaging and spectral analysis, we observed remarkable reduction of autofluorescence of formalin fixed paraffinembedded tissues irradiated with light prior to incubation with immunofluorescent dyes. The technique of photobleaching offers significant improvement in the quality and specificity of immunofluorescence. This has the potential for better techniques for disease diagnosis. PMID:26040118

The paraffin particles were prepared by quenching process after sonicating the solution of paraffin and water at 80 degrees C. The resultant paraffin particles were then used as template for the preparation of macroporous zirconia materials. For this, zirconium normal butoxide (ZNB) modified with triethanolamine (TEA) was first hydrolyzed by water containing the dispersed paraffin particles with the surfactant, Sodium di(2-ethylhexyl) sulfosuccinate. This resulted in the formation of a slurry consisting of hydrolyzed sol and paraffin particles. After centrifugation, a cake packed with hydrated sol and paraffin particles were obtained which was then subjected to heat treatment. The sample obtained after heat treatment contained finely dispersed pores in the size range from 40 nm to 2 microm. Moreover, using the present approach it has also been observed that, change in pore size of zirconia wall is possible with a change in size of the paraffin particles. Thus, the present approach is a novel way of producing porous materials as the particle size of the template could be changed and templates become hard when they were molded as compared to the conventional methods in which there is no change in phase for the templates under 100 degrees C. PMID:17350317

A method of embedding auxiliary information into the digital representation of host data containing noise in the low-order bits. The method applies to digital data representing analog signals, for example digital images. The method reduces the error introduced by other methods that replace the low-order bits with auxiliary information. By a substantially reverse process, the embedded auxiliary data can be retrieved easily by an authorized user through use of a digital key. The modular error embedding method includes a process to permute the order in which the host data values are processed. The method doubles the amount of auxiliary information that can be added to host data values, in comparison with bit-replacement methods for high bit-rate coding. The invention preserves human perception of the meaning and content of the host data, permitting the addition of auxiliary data in the amount of 50% or greater of the original host data.

Contamination of aquatic ecosystems with chlorinated organic contaminants is an increasing toxicological problem. Chlorinated paraffins (CPs) are a class of compounds which are used as fire retardants in paints and as high pressure lubricants and are classified as Priority Toxic Substances under the Canadian Environmental Protection Act (CEPA). As the largest group of high molecular weight chlorinated hydrocarbons produced in Western Europe and North America, CPs have recently come under increased regulatory scrutiny in Canada, the USA, and Europe, because of concerns about their environmental persistence, possible adverse effects on terrestrial and aquatic organisms, and potential carcinogenicity to humans. In both the monitoring of such contamination and the determination of the success of remediation methods, straightforward and inexpensive analytical methodology increases the ease of environmental assessment and facilitates regulatory enforcement. CPs have been considered to be incapable of being analyzed by GC. The authors report the solid phase microextraction (SPME) based analysis of CPs by GC using electron capture (EC) detection. Both synthetic C{sub 10} standards and fractionated commercially derived material have been analyzed by this method in water.

Alterations of BRAF are the most common known genetic aberrations in pediatric gliomas. They frequently are found in pilocytic astrocytomas, where genomic duplications involving BRAF and the poorly characterized gene KIAA1549 create fusion proteins with constitutive B-Raf kinase activity. BRAF V600E point mutations are less common and generally occur in nonpilocytic tumors. The development of BRAF inhibitors as drugs has created an urgent need for robust clinical assays to identify activating lesions in BRAF. KIAA1549-BRAF fusion transcripts have been detected in frozen tissue, however, methods for FFPE tissue have not been reported. We developed a panel of FFPE-compatible quantitative RT-PCR assays for the most common KIAA1549-BRAF fusion transcripts. Application of these assays to a collection of 51 low-grade pediatric gliomas showed 97% sensitivity and 91% specificity compared with fluorescence in situ hybridization or array comparative genomic hybridization. In parallel, we assayed samples for the presence of the BRAF V600E mutation by PCR pyrosequencing. The data further support previous observations that these two alterations of the BRAF, KIAA1549 fusions and V600E point mutations, are associated primarily with pilocytic astrocytomas and nonpilocytic gliomas, respectively. These results show that fusion transcripts and mutations can be detected reliably in standard FFPE specimens and may be useful for incorporation into future studies of pediatric gliomas in basic science or clinical trials. PMID:21884820

Scrapie is a naturally occurring fatal neurodegenerative disease of adult sheep and goats, one of a group of mammalian diseases known as transmissible spongiform encephalopathies or prion diseases. Immunoassays that identify disease-associated prion protein (PrP**Sc) are integral to the diagnosis o...

Fixation techniques preserving morphological fidelity, protein antigenicity and integrity of nucleic acids can have a high impact on both basic and applied biomedical sciences and diagnostic pathology. Different types of mouse tissues were fixed with neutral buffered formalin, ethanol supplemented with acetic acid and modified methacarn (methanol-Carnoy) fixative. The alcohol-fixed samples were processed in an Autotechnicon tissue processor or in an incubator. The preservation of tissue morphology was assessed in all specimens and the immunoreactivity was evaluated with antibodies specific for proteins with nuclear, membrane or cytoplasmic localization. RNA was extracted from all groups of fixed hind limb skeletal muscle specimens and was assessed versus unfixed tissue for preservation of its quantity and quality by amplification of gene-specific fragments of different lengths. Both alcohol-based fixatives preserved the tissue architecture and the specificity of immunoreactivity in excellent quality; the trimming approach did not result in detectable differences. Oligonucleotide fragments of length between 108 and 577 base pairs were amplified from all groups of alcohol-fixed skeletal muscle specimens in amounts comparative to the unfixed muscle tissue. We conclude that both alcohol-based fixatives are an excellent tool for storage of tissue samples designed for immunohistochemical and mRNA expression studies when the access to fresh samples is limited. PMID:22921675

Despite continuous improvement of immunosuppression, small bowel transplantation (SBT) is plagued by a high incidence of acute cellular rejection (ACR) that is frequently intractable. Therefore, there is a need to uncover novel insights that will lead to strategies to achieve better control of ACR. We hypothesized that particular miRNAs provide critical regulation of the intragraft immune response. The aim of our study was to identify miRNAs involved in intestinal ACR. We examined 26 small intestinal mucosal biopsies (AR/NR group; 15/11) obtained from recipients after SBT or multivisceral transplantation. We investigated the expression of 384 mature human miRNAs and 280 mRNAs associated with immune, inflammation and apoptosis processes. We identified differentially expressed 28 miRNAs and 58 mRNAs that characterized intestinal ACR. We found a strong positive correlation between the intragraft expression levels of three miRNAs (miR-142-3p, miR-886-3p and miR-132) and 17 mRNAs including CTLA4 and GZMB. We visualized these miRNAs within cells expressing CD3 and CD14 proteins in explanted intestinal allografts with severe ACR. Our data suggested that miRNAs have a critical role in the activation of infiltrating cells during intestinal ACR. These differences in miRNA expression patterns can be used to identify novel biomarkers and therapeutic targets for immunosuppressive agents. PMID:22026534

The transcriptional response of several cytokines in the spleen of chicken naturally infected by Newcastle Disease velogenic viscerotropic viruses was compared to the responses of atypical velogenic, velogenic neurotropic, and mesogenic strains during the first five days after infection. The ribonuc...

Diagnosis of prion disease is primarily through immunodetection of the infectious agent. Typically, 2 distinct procedures are recommended for a definitive diagnosis with immunohistochemistry and Western blot providing the most information as to the specific isolate in question. In the past these app...

O-6-methylguanine-DNA methyltransferase (MGMT) has been associated with resistance to alkylating agent cancer therapy in Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults. Lower expression or silencing of the MGMT protein by promoter methylation has been reported to improve survival in patients with GBM [1]. This protocol describes bisulfite conversion, methylation sensitive PCR amplification and data analysis/interpretation. This protocol differs from published protocols in that it:•Describes a detailed method to measure MGMT using DNA extracted from solid tumor tissue. We have optimized the DNA extraction by using FFPE tissue blocks that contain greater than 50% tumor tissue, when non-tumor tissue was also present. Performance of this assay is compromised when lower quantities of tumor cells are used as the methylation status of tumor cells is diluted out by methylation status of normal cells.•The measurement of MGMT could be further (enhanced) optimized using a percentage of methylation ration cutoff of 2 as methylated.•The machine specifications detailed here are specific to measuring MGMT from PPFE tumor tissue. PMID:26150933

High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues. PMID:26165823

Recent advances in the diagnostic of myeloproliferative neoplasms (MPNs) discovered CALRETICULIN (CALR) mutations as a major driver in these disorders. In contrast to JAK2 mutations being mainly associated with polycythaemia vera, CALR mutations are only associated with primary myelofibrosis (PMF) and essential thrombocythaemia (ET). CALR mutations are present in the majority of PMF and ET patients lacking JAK2 and MPL mutations. As these CALR mutations are absent from reactive bone marrow (BM) lesions their presence indicates ET or PMF. So far these mutations are detectable only by molecular assays. Their molecular detection is cumbersome because of the great CALR mutation heterogeneity. Therefore, the availability of a simple assay would be of great help. All CALR mutations reported lead to a frameshift generating a new 36 amino-acid C-terminus. We generated a monoclonal antibody (CAL2) to this C-neoterminus by immunizing mice with a representative peptide and compared its performance with Sanger sequencing data in 173 MPNs and other BM diseases. There was a 100% correlation between the molecular and the CAL2 immunohistochemical (IHC) assays. Thus, the detection of CALR mutations by the CAL2 IHC is a specific, sensitive, rapid, simple and low-cost method. PMID:26202929

Interatomic force and energy calculation subroutine to be used with the molecular dynamics simulation code LAMMPS (Ref a.). The code evaluated the total energy and atomic forces (energy gradient) according to a cubic spline-based variant (Ref b.) of the Modified Embedded Atom Method (MEAM) with a additional Stillinger-Weber (SW) contribution.

Online learners often stay located in, and tied to, their communities, kinship networks, households, and workplaces. Institutions providing online education can thus create ties to communities as students draw their learning into networks in which they are already embedded. Frequent interactions across multiple media that are afforded by…

We introduce a model for embedding one network into another, focusing on the case where network A is much bigger than network B. Nodes from network A are assigned to the nodes in network B using an algorithm where we control the extent of localization of node placement in network B using a single parameter. Starting from an unassigned node in network A, called the source node, we first map this node to a randomly chosen node in network B, called the target node. We then assign the neighbors of the source node to the neighborhood of the target node using a random walk based approach. To assign each neighbor of the source node to one of the nodes in network B, we perform a random walk starting from the target node with stopping probability α. We repeat this process until all nodes in network A have been mapped to the nodes of network B. The simplicity of the model allows us to calculate key quantities of interest in closed form. By varying the parameter α, we are able to produce embeddings from very local (α = 1) to very global (α --> 0). We show how our calculations fit the simulated results, and we apply the model to study how social networks are embedded in geography and how the neurons of C. Elegans are embedded in the surrounding volume.

A review of literature and two surveys, one of college students and one of a random sample of adults, were used to examine four aspects of media embedded interactions (social behavior in front of a TV or radio): their functions, their environment, their effects, and the reactions of the interactants to them. Television is seen as performing a…

An indefinite metric polyhedron is a triple (X, T, g) where X is a topological space, T is a simplicial triangulation of X with edge set E, and g is a function from E to the reals. g assigns to each k-dimensional simplex S a unique quadratic form on Rk, denoted by G(S). An indefinite metric polyhedron is called a Euclidean polyhedron if the form G(S) is positive definite for every simplex S. Rpq denotes R p + q endowed with the inner product of signature (p, q). Our first result is that every compact n-dimensional indefinite metric polyhedron with vertex set V admits a simplicial isometric embedding into Rqq where q = max{d, 2n + 1} and d = max{deg(v) | v is in V}. We can use the compact case to extend to the non-compact case, but only if we assume that d = max{deg(v) | v is in V} is less than infinity. Specifically, every (non-compact) indefinite metric polyhedron admits a simplicial isometric embedding into Rpp where p = 2q(d3 - d2 + d + 1) and q and d are defined as above. Finally we use results of Akopyan and Greene to prove that every n-dimensional indefinite metric polyhedron admits a piecewise linear isometric embedding into Rn2n. In Chapter 2 we prove that every short (1-Lipschitz) map from an n-dimensional Euclidean polyhedron into EN is epsilon close to a pl isometric embedding (for anyepsilon > 0) provided N ≥ 3n. We can relax the dimensionality of the Euclidean space to 2n + 1 if we allow our map to be continuous instead of pl. These results are extensions of a result due to Akopyan. We provide a detailed proof of Akopyan's Theorem, as the only currently available proof is in Russian. The remaining results in this work are applications of our continuous isometric embedding theorem above. This result is used to prove that every Pro-Euclidean space of rank at most n admits an isometric embedding into E2n + 1. The result, as well as a theorem due to Bridson, also allows for an approximate isometric embedding theorem for geodesic metric spaces with

A case of acute paraffin oil-induced pneumonia due to accidental inhalation by a fire-eater of kerdane, a petroleum derivative is reported. The symptoms and course of respiratory manifestations of acute paraffin oil poisoning are reviewed. The physical properties of the petroleum derivative inhaled account for the pathogenesis of the pneumonia. Pulmonary lesions, usually fully reversible, result from the joint effects of an inflammatory phase with exudate and a proliferative phase. PMID:3406615

A thermal stimulation process that generates downhole heat is presently being used to stimulate oil wells in the Gulf of Mexico. The crude produced from these shallow wells is highly paraffinic. Previous attempts to stimulate these wells with paraffin solvents and acid systems have been unsuccessful. The in situ heat process has yielded tenfold production rate increases and payout times of less than one week.

A thermal stimulation process that generates downhole heat currently is being used to stimulate oil wells in the Gulf of Mexico. The crude produced from these shallow wells is highly paraffinic. Previous attempts to stimulate these wells with paraffin solvents and acid systems have been unsuccessful. The in-situ heat process has yielded 10-fold production rate increases and payout times of less than 1 week.

Methane is sparingly soluble in water, resulting in a slow reaction rate in the denitrifying anaerobic methane oxidation (DAMO) process. The slow rate limits the feasibility of research to examine the interaction between the DAMO and the anaerobic ammonium oxidation (Anammox) process. In this study, optimized 5 % (v/v) paraffin oil was added as a second liquid phase to improve methane solubility in a reactor containing DAMO and Anammox microbes. After just addition, methane solubility was found to increase by 25 % and DAMO activity was enhanced. After a 100-day cultivation, the paraffin reactor showed almost two times higher consumption rates of NO3 (-) (0.2268 mmol/day) and NH4 (+) (0.1403 mmol/day), compared to the control reactor without paraffin oil. The microbes tended to distribute in the oil-water interface. The quantitative (q) PCR result showed the abundance of gene copies of DAMO archaea, DAMO bacteria, and Anammox bacteria in the paraffin reactor were higher than those in the control reactor after 1 month. Fluorescence in situ hybridization revealed that the percentages of the three microbes were 55.5 and 77.6 % in the control and paraffin reactors after 100 days, respectively. A simple model of mass balance was developed to describe the interactions between DAMO and Anammox microbes and validate the activity results. A mechanism was proposed to describe the possible way that paraffin oil enhanced DAMO activity. It is quite clear that paraffin oil enhances not only DAMO activity but also Anammox activity via the interaction between them; both NO3 (-) and NH4 (+) consumption rates were about two times those of the control. PMID:26036704

This investigation studied the inclusion of various additives to paraffin wax for use in a hybrid rocket motor. Some of the paraffin-based fuels were doped with various percentages of LiAlH4 (up to 10%). Addition of LiAlH4 at 10% was found to increase regression rates between 7 - 10% over baseline paraffin through tests in a gaseous oxygen hybrid rocket motor. Mass burn rates for paraffin grains with 10% LiAlH4 were also higher than those of the baseline paraffin. RDX was also cast into a paraffin sample via a novel casting process which involved dissolving RDX into dimethylformamide (DMF) solvent and then drawing a vacuum on the mixture of paraffin and RDX/DMF in order to evaporate out the DMF. It was found that although all DMF was removed, the process was not conducive to generating small RDX particles. The slow boiling generated an inhomogeneous mixture of paraffin and RDX. It is likely that superheating the DMF to cause rapid boiling would likely reduce RDX particle sizes. In addition to paraffin/LiAlH4 grains, multi-walled carbon nanotubes (MWNT) were cast in paraffin for testing in a hybrid rocket motor, and assorted samples containing a range of MWNT percentages in paraffin were imaged using SEM. The fuel samples showed good distribution of MWNT in the paraffin matrix, but the MWNT were often agglomerated, indicating that a change to the sonication and mixing processes were required to achieve better uniformity and debundled MWNT. Fuel grains with MWNT fuel grains had slightly lower regression rate, likely due to the increased thermal conductivity to the fuel subsurface, reducing the burning surface temperature.

The aim of the present study was to develop an immunohistochemical method (IHC) for detection of Lawsonia intracellularis (L. intracellularis) in formalin-fixed, paraffinembeddedsections of intestines from pigs and to implement this method in differential diagnosis of swine diseases with diarrhea in postweaning pigs. The study was conducted on 165 sections of intestines (ileum, caecum and colon) collected from 76 pigs, representing 42 Polish pig farms. The animals included in the analysis suffered from diarrhea, with bloody or grey to brown feces, and were suspected of porcine proliferative enteropathy (PPE). Sections of intestines were analyzed for the presence of L. intracellularis by polymerase chain reaction (PCR) and IHC. Among 165 intestinal samples from pigs with diarrhea, L. intracellularis DNA was detected by PCR in 33 (20.0%) samples. In this group, 30 samples (18.2% of all the samples tested) were also found positive in IHC, while only 3 (1.8%) were IHC-negative. One hundred thirty-two (80.0%) samples were negative in both tests. The PCR- and IHC-positive samples originated from 11 pigs, 4- to 20-week old, from 8 farms. L. intracellularis antigen was visualized by IHC mostly in intestinal crypts and/or in mononuclear cells of the lamina propria). The positive signal in epithelial cells was observed close to the luminal borders, creating typical specifically stained rims around the crypt lumina. The results of the present study further confirm the usefulness of IHC in the detection of L. intracellularis antigen in the intestinal tissues. PMID:22439321

Polar monomer acrylic acid (AA) was used to modify paraffin in order to improve the latent heat of paraffin as phase change materials. The composition and sequence structure of the grafted products were characterized by FTIR, 13C NMR, 1H NMR and GPC analysis, and the thermal properties of paraffin-g-AA were investigated. It was found that AA was confirmed to be grafted onto the molecular chain of paraffin successfully. The mechanism of free radical grafting of AA may be only monomeric grafts. At low grafting ratio, the structure B can be mainly formed as a result of the radical coupling termination; while at the high grafting ratio, structure A was the primary structure as a result of the radical chain growth process. The number-average molecular weight of the grafted samples increased at first but leveled off with increasing grafting ratio, while the weight-average molecular weight increased gradually. The latent heat capacity of the grafted paraffin can be improved obviously at low grafting ratio due to the formation of structure B.

This paper describes the operational concept of the Embedded Instrumentation Systems Architecture (EISA) that is being developed for Test and Evaluation (T&E) applications. The architecture addresses such future T&E requirements as interoperability, flexibility, and non-intrusiveness. These are the ultimate requirements that support continuous T&E objectives. In this paper, we demonstrate that these objectives can be met by decoupling the Embedded Instrumentation (EI) system into an on-board and an off-board component. An on-board component is responsible for sampling, pre-processing, buffering, and transmitting data to the off-board component. The latter is responsible for aggregating, post-processing, and storing test data as well as providing access to the data via a clearly defined interface including such aspects as security, user authentication and access control. The power of the EISA architecture approach is in its inherent ability to support virtual instrumentation as well as enabling interoperability with such important T&E systems as Integrated Network-Enhanced Telemetry (iNET), Test and Training Enabling Architecture (TENA) and other relevant Department of Defense initiatives.

Most foods from plant origin usually contain 1-10 mg/kg (dry weight) of non-resolved isomeric alkanes in the range of the n-alkanes C 20-C 50 which are assumed to be residues from mineral oil products (in addition to the natural paraffins). In edible vegetable oils, concentrations may exceed 100 mg/kg. Since it was suspected that this contamination was mostly of environmental origin, particulate matter from air was analysed for the same range of paraffins. In a road tunnel, around 5 μg/m 3 of such paraffins were found, corresponding to about 3% of the fine dust (PM10). The composition corresponded to that found in the particulate matter from the exhaust of diesel engines, which in turn largely corresponded to engine (lubricating) oil. In Swiss cities, the C 20-C 50 mineral paraffins in the PM10 from ambient air amounted to 0.1-1.5 μg/m 3 (about 1% of the dust) and seemed to primarily originate from incomplete combustion of heating and diesel oil, lubricating oil, and road tar debris. On the countryside, the concentrations were around 0.03 μg/m 3 (0.3% of the dust). Soil contained 0.5-10 mg/kg of these paraffins. The similarity of the molecular weight (volatility) distribution suggests that the food contamination with paraffins, is mostly from the air. A substantial proportion probably consists of lubricating oil. If this hypothesis is confirmed, measures should be investigated to reduce this contamination.

Relative rate constants of coke formation from saturated hydrocarbons, olefins, and acetylenes during steam cracking of naphtha at 810 C were determined by application of [sup 14]C-labeled compounds. The range of hydrocarbons investigated comprises 20 representatives of paraffins, from methane to hexadecane, and of naphthenes, from cyclopentane to hydroanthracene, and 20 olefins from ethene to styrene, including cyclo- and diolefins, and five acetylenes. Carbonaceous deposits in pyrolysis reactors from steel and quartz as well as in the transfer line exchange (TLE) section were determined separately.

The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues. Method correlation with sequencing showed 96% mutation-wise agreement and 99% chip-wise agreement. We furthermore observed 100% agreement (113/113) of the most prevalent TP53 mutations. Workflow reproducibility was 96.8% across 8 samples, with 2 operators, 2 reagent lots and 2 instruments. Section-to-section reproducibility was 100% for each sample across a 60 μm region of the FFPE block from ovarian tumors. These data indicate that the AmpliChip p53 Research Test is an accurate and reproducible method for detecting mutations in TP53 from archival FFPE human ovarian specimens. PMID:26125596

Drying of the tissue section, partial or total, during immunostaining negatively affects both the staining of tissue antigens and the ability to remove previously deposited antibody layers, particularly during sequential rounds of de-staining and re-staining for multiple antigens. The cause is a progressive loss of the protein-associated water up to the removal of the non-freezable water, a step which abolishes the immunoavailability of the epitope. In order to describe and prevent these adverse effects, we tested, among other substances, sugars, which are known to protect unicellular organisms from freezing and dehydration, and stabilize drugs and reagents in solid state form in medical devices. Disaccharides (lactose, sucrose) prevented the air drying-induced antigen masking and protected tissue-bound antigens and antibodies from air drying-induced damage. Complete removal of the bound antibody layers by chemical stripping was permitted if lactose was present during air drying. Lactose, sucrose and other disaccharides prevent air drying artifacts, allow homogeneous, consistent staining and the reuse of formalin-fixed, paraffin-embedded tissue sections for repeated immunostaining rounds by guaranteeing constant staining quality in suboptimal hydration conditions. PMID:26487185

Frozen-Density-Embedding Theory (FDET) is a formalism to obtain the upper bound of the ground-state energy of the total system and the corresponding embedded wavefunction by means of Euler-Lagrange equations [T. A. Wesolowski, Phys. Rev. A 77(1), 012504 (2008)]. FDET provides the expression for the embedding potential as a functional of the electron density of the embedded species, electron density of the environment, and the field generated by other charges in the environment. Under certain conditions, FDET leads to the exact ground-state energy and density of the whole system. Following Perdew-Levy theorem on stationary states of the ground-state energy functional, the other-than-ground-state stationary states of the FDET energy functional correspond to excited states. In the present work, we analyze such use of other-than-ground-state embedded wavefunctions obtained in practical calculations, i.e., when the FDET embedding potential is approximated. Three computational approaches based on FDET, that assure self-consistent excitation energy and embedded wavefunction dealing with the issue of orthogonality of embedded wavefunctions for different states in a different manner, are proposed and discussed.

Pre-embedding nanogold silver and gold intensification methods involve immunoreactions with nanogold-labeled antibodies and intensification of the nanogold particles before embedding and ultrathin sectioning. These highly sensitive methods show good resolution and ultrastructural preservation. They also are useful for simultaneous observation of immunolabeled cells under light and electron microscopes, and for three-dimensional immunoelectron microscopic analyses. Silver intensification is usually superior for immunolabeling. On the other hand, ultrastructural preservation is better when gold intensification is used. In this chapter, we introduce pre-embedding nanogold silver and gold intensification procedures for use primarily with cultured cells. PMID:27515087

Stellar clusters are born embedded within giant molecular clouds (GMCs) and during their formation and early evolution are often only visible at infrared wavelengths, being heavily obscured by dust. Over the past 15 years advances in infrared detection capabilities have enabled the first systematic studies of embedded clusters in galactic molecular clouds. In this article we review the current state of empirical knowledge concerning these extremely young protocluster systems. From a survey of the literature we compile the first extensive catalog of galactic embedded clusters. We use the catalog to construct the mass function and estimate the birthrate for embedded clusters within 2 kpc of the sun. We find that the embedded cluster birthrate exceeds that of visible open clusters by an order of magnitude or more indicating a high infant mortality rate for protocluster systems. Less than 4-7% of embedded clusters survive emergence from molecular clouds to become bound clusters of Pleiades age. The vast majority (90%) of stars that form in embedded clusters form in rich clusters of 100 or more members with masses in excess of 50 M⊙. Moreover, observations of nearby cloud complexes indicate that embedded clusters account for a significant (70-90%) fraction of all stars formed in GMCs. We review the role of embedded clusters in investigating the nature of the initial mass function (IMF) that, in one nearby example, has been measured over the entire range of stellar and substellar mass, from OB stars to substellar objects near the deuterium burning limit. We also review the role embedded clusters play in the investigation of circumstellar disk evolution and the important constraints they provide for understanding the origin of planetary systems. Finally, we discuss current ideas concerning the origin and dynamical evolution of embedded clusters and the implications for the formation of bound open clusters.

Nuclide leaching models based on mass transfer theory are reviewed and evaluated to analyze the leaching test results of simulated and real paraffin waste from Korean nuclear power plants (NPPs). An empirical model (EM), bulk diffusion model (BDM), coupled diffusion/dissolution model (CDDM), shrinking core model (SCM), modified SCM (MSCM), and uniform reaction model (URM) are selected for comparison. In case of simulated paraffin waste form, the experimental results are satisfactorily explained by the SCM which is based on a diffusion-controlled dissolution reaction. Leaching behavior of real paraffin waste form is well predicted by URM that considers inter-aggregated porous medium and intra-aggregated porous medium separately. If real paraffin waste forms are manufactured with relatively uniform composition, their leaching behaviors are expected to be similar to those of simulated paraffin waste forms. PMID:12169419

In this study, biosurfactant-producing strain N2 and non-biosurfactant producing stain KB18 were used to investigate the effects of microbial treatment on the prevention and removal of paraffin deposits on stainless steel surfaces. Strain N2, with a biosurfactant production capacity, reduced the contact angle of stainless steel to 40.04°, and the corresponding adhesion work of aqueous phase was decreased by 26.5 mJ/m(2). By contrast, KB18 could only reduce the contact angle to 50.83°, with a corresponding 7.6 mJ/m(2) decrease in the aqueous phase work adhesion. The paraffin removal test showed that the paraffin removal efficiencies of strain N2 and KB18 were 79.0% and 61.2%, respectively. Interestingly, the N2 cells could attach on the surface of the oil droplets to inhibit droplets coalescence. These results indicate that biosurfactant-producing strains can alter the wettability of stainless steel and thus eliminate paraffin deposition. PMID:22989649

This report presents an analysis of steam heating of a two-layer tri-n-butyl phosphate (TBP)/n-paraffin-nitric acid mixture.The purpose of this study is to determine if the degree of mixing provided by the steam jet or by bubbles generated by the TBP/nitric acid reaction is sufficient to prevent a runaway reaction.

The continued evolution of military munitions and armor on the battlefield, as well as the insurgent use of improvised explosive devices, has led to embedded fragment wounds containing metal and metal mixtures whose long-term toxicologic and carcinogenic properties are not as yet known. Advances in medical care have greatly increased the survival from these types of injuries. Standard surgical guidelines suggest leaving embedded fragments in place, thus individuals may carry these retained metal fragments for the rest of their lives. Nursing professionals will be at the forefront in caring for these wounded individuals, both immediately after the trauma and during the healing and rehabilitation process. Therefore, an understanding of the potential health effects of embedded metal fragment wounds is essential. This review will explore the history of embedded fragment wounds, current research in the field, and Department of Defense and Department of Veterans Affairs guidelines for the identification and long-term monitoring of individuals with embedded fragments. PMID:25222538

Solid fuels for hybrid rockets were characterized in the framework of a research project aimed to develop a new generation of solid fuels, combining at the same time good mechanical and ballistic properties. Original techniques were implemented in order to improve paraffin-based fuels. The first strengthening technique involves the use of a polyurethane foam (PUF); a second technique is based on thermoplastic polymers mixed at molecular level with the paraffin binder. A ballistic characterization of paraffin-based hybrid rocket solid fuels was performed, considering pure wax-based fuels and fuels doped with suitable metal additives. Nano-Al powders and metal hydrides (magnesium hydride (MgH2), lithium aluminum hydride (LiAlH4 )) were used as fillers in paraffin matrices. The results of this investigation show a strong correlation between the measured viscosity of the melted paraffin layer and the regression rate: a decrease of viscosity increases the regression rate. This trend is due to the increasing development of entrainment phenomena, which strongly increase the regression rate. Addition of LiAlH4 (mass fraction 10%) can further increase the regression rate up to 378% with respect to the pure HTPB regression rate, taken as baseline reference fuel. The highest regression rates were found for the Solid Wax (SW) composition, added with 5% MgH2 mass fraction; at 350 kg/(m2s) oxygen mass flux, the measured regression rate, averaged in space and time, was 2.5 mm/s, which is approximately five times higher than that of the pure HTPB composition. Compositions added with nanosized aluminum powders were compared with those added with MgH2, using gel or solid wax.

Language use has a public face that is as important to study as the private faces under intensive psycholinguistic study. In the domain of phonology, public use of speech must meet an interpersonal “parity” constraint if it is to serve to communicate. That is, spoken language forms must reliably be identified by listeners. To that end, language forms are embodied, at the lowest level of description, as phonetic gestures of the vocal tract that lawfully structure informational media such as air and light. Over time, under the parity constraint, sound inventories emerge over communicative exchanges that have the property of sufficient identifiability. Communicative activities involve more than vocal tract actions. Talkers gesture and use facial expressions and eye gaze to communicate. Listeners embody their language understandings, exhibiting dispositions to behave in ways related to language understanding. Moreover, linguistic interchanges are embedded in the larger context of language use. Talkers recruit the environment in their communicative activities, for example, in using deictic points. Moreover, in using language as a “coordination device,” interlocutors mutually entrain. PMID:21243080

Impact damage in graphite/epoxy laminates was characterized and transient strain history during impact was correlated. The material investigated was AS-4/3501-6 graphite/epoxy. Eight-ply and sixteen-ply quasi-isotropic laminates of 45/0/-45/90 sub s and 45/0/-45/90 sub 2s layups were fabricated with strain gages embedded between plies during the strain gages and leads from the highly conductive graphite fibers. The specimens were circular plates 12.7 cm (5 in.) in diameter and clamped along their circumference. The specimens were impacted with a 185 gm impactor, dropped from heights of 1.20 m and 1.65 m. An accelerometer was attached to the back surface of the specimen opposite the impact point and was used to trigger the recording instrumentation. The transient strain data were recorded with an eight channel waveform digitizer capable of sampling data at 0.5 microsec intervals. The data were stored, processed, and plotted by means of a microcomputer. Transient strain data were correlated with results from ultrasonic inspection of the specimens.

Layered Manufacturing can be applied to build ``smart'' parts with sensors, integrated circuits, and actuators placed within the component. Embedded sensors can be used to gain data for validating or improving designs during the prototype stage or to obtain information on the performance and structural integrity of components in service. Techniques for embedding fiber optic sensors in metals, polymers, and ceramics have been investigated. Embedding optical fibers into metals is especially challenging because engineering alloys tend to exhibit high melting temperatures. In the present research an embedding sequence was developed capable of embedding fiber sensors into parts made of metal alloys with high melting temperatures. Fiber Bragg Grating (FBG) sensors were selected as the most promising sensor candidate. The embedded FBG sensors were characterized for temperature and strain measurements. The embedded FBG sensors in nickel and stainless steel provided high sensitivity, good accuracy, and high temperature capacity for temperature measurements. Temperature sensitivity approximately 100% higher than that of bare FBGs was demonstrated. For strain measurements, the sensors embedded in metal and polyurethane yielded high sensitivity, accuracy, and linearity. The sensitivity of the embedded FBGs was in good agreement with that of bare FBGs. Moreover, a decoupling technique for embedded FBG sensors was developed to separate temperature and strain effects. The embedded FBG sensors were used to monitor the accumulation of residual stresses during the laser- assisted Layered Manufacturing, to measure the strain field in layered materials, to measure pressure, and to monitor temperature and strain simultaneously. New techniques have been developed for temperature and strain measurements of rotating components with FBG sensors embedded or attached to the surface. Tunable laser diodes were incorporated into the sensing system for monitoring the Bragg grating wavelength

Embedded fiducials are provided in optical surfaces and a method for embedding the fiducials. Fiducials, or marks on a surface, are important for optical fabrication and alignment, particularly when individual optical elements are aspheres. Fiducials are used during the course of the polishing process to connect interferometric data, and the equation describing the asphere, to physical points on the optic. By embedding fiducials below the surface of the optic and slightly outside the clear aperture of the optic, the fiducials are not removed by polishing, do not interfere with the polishing process, and do not affect the performance of the finished optic.

Embedded fiducials are provided in optical surfaces and a method for embedding the fiducials. Fiducials, or marks on a surface, are important for optical fabrication and alignment, particularly when individual optical elements are aspheres. Fiducials are used during the course of the polishing process to connect interferometric data, and the equation describing the asphere, to physical points on the optic. By embedding fiducials below the surface of the optic and slightly outside the clear aperture of the optic, the fiducials are not removed by polishing, do not interfere with the polishing process, and do not affect the performance of the finished optic.

Fluorescent in situ hybridization (FISH) is a technique which complements conventional cytogenetic banding analysis by allowing the evaluation of cells in interphase as well as metaphase. This technique has been used to study air-dried peripheral blood and bone marrow aspirate smears. We have applied the FISH technique to study routinely processed sections of bone marrow aspirate clot and decalcified core biopsy specimens, fixed in either formalin or B5 and embedded in paraffin. We evaluated 28 specimens (8 aspirate clot and 20 core biopsy sections) for chromosome 8 copy number, studied previously by conventional cytogenetics, and found the following distribution: 15 with disomy, 11 with trisomy, and 2 with tetrasomy. Using a chromosome 8 alpha-satellite probe, we detected fluorescent hybridization signals in 18 of 28 specimens (64%); 6 of 8 (75%) aspirate clot sections, and 12 of 20 (60%) core biopsy sections. Ten of 13 (77%) B5-fixed and 8 of 15 (53%) formalin-fixed specimens had hybridizing signals. Specimen age was a significant factor; 10 of 11 (91%) specimens processed within the last 6 months showed signals, in contrast with 8 of 17 (47%) specimens older than 6 months. In the positive specimens, 200 cells were analyzed in areas where individual cells could be identified. In the disomic specimens, two signals per cell were seen in 34 to 66% of the cells. Rare cells (0-2%) with three signals were detected. In the trisomic specimens, three signals per cell were seen in 19 to 46% of the cells. In the tetrasomic specimens, four signals per cell were seen in 15 to 25% of the cells. We conclude that the FISH technique may be useful in the detection of numerical chromosomal abnormalities such as trisomy and tetrasomy 8 in routinely processed bone marrow aspirate clot and decalcified core biopsy sections. Images Figure 1 Figure 2 Figure 3 PMID:7992836

{sup 74}Ge nanocrystals are formed in a sapphire matrix by ion implantation followed by damage. Embedded nanocrystals experience large compressive stress relative to bulk, as embedded in sapphire melt very close to the bulk melting point (Tm = 936 C) whereas experience considerably lower stresses. Also, in situ TEM reveals that nanocrystals ion-beam-synthesized nanocrystals embedded in silica are observed to be spherical and measured by Raman spectroscopy of the zone center optical phonon. In contrast, reveals that the nanocrystals are faceted and have a bi-modal size distribution. Notably, the matrix remains crystalline despite the large implantation dose and corresponding thermal annealing. Transmission electron microscopy (TEM) of as-grown samples those embedded in silica exhibit a significant melting point hysteresis around T{sub m}.

In this work is considered embedding theory, a theory in which independent variables which describe gravity are functions of the space-time embedding into a ten-dimensional pseudo-Euclidean space. Neother's theorem is used to find conservation laws for energy and angular momentum as a result from the action's invariance in relation to the rotation and translation of the system. The form of these conservation laws and their consequences depending on the different formulations of embedding theory is discussed. It is also analyzed a transition from embedding theory to a field theory in a flat space-time with a number of dimensions greater than four. The same procedure is followed in this case to find conservation laws, resulting in the solution of the problem of time present in Einstein's theory of general relativity.

Introduction: Leptospirosis is a zoonotic disease affecting mainly to low income human population. Acute leptospiral infection during pregnancy has been associated with spontaneous abortion and fetal death during the first trimester and the abortion may occur as consequence of systemic failure. Objective: To estimate the frequency of Leptospira interrogans infection in women with spontaneous abortion in the state of Yucatan, Mexico. Methods: A cross sectional study on women with spontaneous abortion was conducted. Serum samples were tested for Leptospirosis by the microaglutination test, to estimate the frequency of the infecting serovar. The indirect ELISA IgM was used to detect recent infection by L. interrogans. DNA was extracted from paraffin-embedded tissue of placenta for PCR detection of L. interrogans. Results: Overall frequency of infection with L. interrogans in the 81 women with abortion was 13.6%. Five of the 12 serovars evaluated were found and included. Two of the 11 women with abortion and positive to microaglutination test were also positive to the ELISA IgM test. None samples were positive for PCR Leptospira diagnosis. Conclusion: two women could be associated with spontaneous abortion due to leptospirosis, because they showed antibodies against L. interrogans in the microaglutination test and ELISA IgM assays. Differences between regions were found with respect to the prevalences of lesptospirosis. PMID:27226658

It is shown how to obtain conformal blocks from embedding space with the help of the operator product expansion. The minimal conformal block originates from scalar exchange in a four-point correlation function of four scalars. All remaining conformal blocks are simple derivatives of the minimal conformal block. With the help of the orthogonality properties of the conformal blocks, the analytic conformal bootstrap can be implemented directly in embedding space, leading to a Jacobi-like definition of conformal field theories.

Liquid paraffin is a highly refined petroleum derivative commonly used medicinally as an oral laxative in Lesotho. We present the case of a 22-year-old Basotho woman admitted under the care of gynaecology in a rural hospital in Lesotho. She was inadvertently administered 10 mL of intravenous liquid paraffin. There were no immediate complications. After 48 h, the patient became unwell with frank haemoptysis and features of systemic inflammation. A chest X-ray demonstrated new bilateral pulmonary infiltrates. She made a full clinical and radiological recovery with a 5-day course of high-dose oral prednisolone and broad-spectrum antibiotics. She was discharged home in a stable condition. PMID:26791127

Paraffin and asphaltene related problems including solid deposits, stabilization of emulsions and sludge production continue to plaque the oil and gas industry. Condensates and refined aromatic solvents are popular treatments for dissolving and/or controlling paraffin and asphaltene related problems. These treating fluids are typically used as a quick fix with little regard for formation damage consequences and long term effectiveness. Testing, blending and refining of unique condensate feedstocks has resulted in unique natural multi-component hydrocarbon solvents. These unique solvents will dissolve a broad carbon number spectrum of organic deposits and keep them in solution under extreme conditions. These natural solvents maximize solvency, demulsifying properties and natural wettibility tendencies without the addition of chemical additives. Those natural solvents offer economic alternatives and enhancement to common treatment practices including condensate treatments, hot oiling, chemical treatments, stimulation and production treatments.

A simple and low cost approach was developed to fabricate a superhydrophobic surface on copper foil. The oxidation and etching of the copper foil surface were promoted in NH4HCO3 solution using a water and ethanol admixture as a component solvent. After 28 h in this solution, a hydrophilic rough surface structure was obtained on the copper foil surface. With modification using a paraffin wax coating, the hydrophilic rough copper surface changed to become hydrophobic or superhydrophobic. The surface morphology and wettability were characterized by scanning electron microscopy (SEM) and contact angle measurements, respectively. When the optimum concentration of paraffin wax was about 2 g L-1, its water contact angle could reach about 152 ± 1.5° and its sliding angle was around 7°. The formation mechanism of the rough copper surface was also explored in detail. Both the experimental process and the material are environmentally friendly.

A standardized workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI imaging MS) is a prerequisite for the routine use of this promising technology in clinical applications. We present an approach to develop standard operating procedures for MALDI imaging MS sample preparation of formalin-fixed and paraffin-embedded (FFPE) tissue sections based on a novel quantitative measure of dataset quality. To cover many parts of the complex workflow and simultaneously test several parameters, experiments were planned according to a fractional factorial design of experiments (DoE). The effect of ten different experiment parameters was investigated in two distinct DoE sets, each consisting of eight experiments. FFPE rat brain sections were used as standard material because of low biological variance. The mean peak intensity and a recently proposed spatial complexity measure were calculated for a list of 26 predefined peptides obtained by in silico digestion of five different proteins and served as quality criteria. A five-way analysis of variance (ANOVA) was applied on the final scores to retrieve a ranking of experiment parameters with increasing impact on data variance. Graphical abstract MALDI imaging experiments were planned according to fractional factorial design of experiments for the parameters under study. Selected peptide images were evaluated by the chosen quality metric (structure and intensity for a given peak list), and the calculated values were used as an input for the ANOVA. The parameters with the highest impact on the quality were deduced and SOPs recommended. PMID:27485623

Formation damage caused by the precipitation and deposition of paraffin or asphaltene particles has been a recurrent problem in the production of crude oil. A number of well established oilfield operations have been found to aggravate these organic deposition problems. Laboratory testing of crudes and chemical additives has led to a number of solutions to these problems. Case history information on testing, chemical application, and subsequent field results are presented.

Core-shell structures of oxide nanoparticles having a high dielectric constant, and organic shells with large breakdown field are attractive candidates for large electrical energy storage applications. A high growth temperature, however, is required to obtain the dielectric oxide nanoparticles, which affects the process of core-shell formation and also leads to poor control of size, shape, and size-distribution. In this communication, we report a new synthetic process to grow core-shell nanoparticles by means of an experimental method that can be easily adapted to synthesize core-shell structures from a variety of inorganic-organic or inorganic-inorganic materials. Monodisperse and spherical TiO2 nanoparticles were produced at room temperature as a collimated cluster beam in the gas phase using a cluster-deposition source and subsequently coated with uniform paraffin nanoshells using in situ thermal evaporation, prior to deposition on substrates for further characterization and device processing. The paraffin nanoshells prevent the TiO2 nanoparticles from contacting each other and also act as a matrix in which the volume fraction of TiO2 nanoparticles was varied by controlling the thickness of the nanoshells. Parallel-plate capacitors were fabricated using dielectric core-shell nanoparticles having different shell thicknesses. With respect to the bulk paraffin, the effective dielectric constant of TiO2-paraffin core-shell nanoparticles is greatly enhanced with a decrease in the shell thickness. The capacitors show a minimum dielectric dispersion and low dielectric losses in the frequency range of 100 Hz-1 MHz, which are highly desirable for exploiting these core-shell nanoparticles for potential applications. PMID:20359188

Using isoreticular chemistry allows the design and construction of a new rare-earth metal (RE) fcu-MOF with a suitable aperture size for practical steric adsorptive separations. The judicious choice of a relatively short organic building block, namely fumarate, to bridge the 12-connected RE hexanuclear clusters has afforded the contraction of the well-defined RE-fcu-MOF triangular window aperture, the sole access to the two interconnected octahedral and tetrahedral cages. The newly constructed RE (Y(3+) and Tb(3+)) fcu-MOF analogues display unprecedented total exclusion of branched paraffins from normal paraffins. The resultant window aperture size of about 4.7 Å, regarded as a sorbate-size cut-off, enabled a complete sieving of branched paraffins from normal paraffins. The results are supported by collective single gas and mixed gas/vapor adsorption and calorimetric studies. PMID:26429515

In this paper, we propose a texture representation framework to map local texture patches into a low-dimensional texture subspace. In natural texture images, textons are entangled with multiple factors, such as rotation, scaling, viewpoint variation, illumination change, and non-rigid surface deformation. Mapping local texture patches into a low-dimensional subspace can alleviate or eliminate these undesired variation factors resulting from both geometric and photometric transformations. We observe that texture representations based on subspace embeddings have strong resistance to image deformations, meanwhile, are more distinctive and more compact than traditional representations. We investigate both linear and non-linear embedding methods including Principle Component Analysis (PCA), Linear Discriminant Analysis (LDA), and Locality Preserving Projections (LPP) to compute the essential texture subspace. The experiments in the context of texture classification on benchmark datasets demonstrate that the proposed subspace embedding representations achieve the state-of-the-art results while with much fewer feature dimensions. PMID:23710105

In this paper, we propose a texture representation framework to map local texture patches into a low-dimensional texture subspace. In natural texture images, textons are entangled with multiple factors, such as rotation, scaling, viewpoint variation, illumination change, and non-rigid surface deformation. Mapping local texture patches into a low-dimensional subspace can alleviate or eliminate these undesired variation factors resulting from both geometric and photometric transformations. We observe that texture representations based on subspace embeddings have strong resistance to image deformations, meanwhile, are more distinctive and more compact than traditional representations. We investigate both linear and non-linear embedding methods including Principle Component Analysis (PCA), Linear Discriminant Analysis (LDA), and Locality Preserving Projections (LPP) to compute the essential texture subspace. The experiments in the context of texture classification on benchmark datasets demonstrate that the proposed subspace embedding representations achieve the state-of-the-art results while with much fewer feature dimensions. PMID:23710105

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM. PMID:23481606

Transducers sensitive to rates of change of temperature embedded in integrated circuits and discrete electronic components damaged by overheating, according to proposal. Used to detect onset of rapid heating and to trigger shutoffs of power or other corrective actions before temperatures rise beyond safe limits. Sensors respond fast and reliably to incipient overheating because they are in direct thermal contact with vulnerable circuit elements.

... is a solid waxy substance used to make candles and other items. This article discusses what may ... Some arthritis bath/spa treatments Some candles Some waxes Note: This list may not be all-inclusive.

This research presents a physical-mathematical model for the combustion of liquefying fuels in hybrid combustors, accounting for blowing effect on the heat transfer. A particular attention is given to a paraffin/nitrous oxide hybrid system. The use of a paraffin fuel in hybrid propulsion has been considered because of its much higher regression rate enabling significantly higher thrust compared to that of common polymeric fuels. The model predicts the overall regression rate (melting rate) of the fuel and the different mechanisms involved, including evaporation, entrainment of droplets of molten material, and mass loss due to melt flow on the condensed fuel surface. Prediction of the thickness and velocity of the liquid (melt) layer formed at the surface during combustion was done as well. Applying the model for an oxidizer mass flux of 45 kg/(s m2) as an example representing experimental range, it was found that 21% of the molten liquid undergoes evaporation, 30% enters the gas flow by the entrainment mechanism, and 49% reaches the end of the combustion chamber as a flowing liquid layer. When increasing the oxidizer mass flux in the port, the effect of entrainment increases while that of the flowing liquid layer along the surface shows a relatively lower contribution. Yet, the latter is predicted to have a significant contribution to the overall mass loss. In practical applications it may cause reduced combustion efficiency and should be taken into account in the motor design, e.g., by reinforcing the paraffin fuel with different additives. The model predictions have been compared to experimental results revealing good agreement.

Naval Petroleum Reserve No. 3 (NPR-3), also known as Teapot Dome, is a government-owned oil field in Natrona County, Wyoming. It is an asymmetrical anticline located on the western edge of the Powder River Basin, just south of the Salt Creek Anticline. Production started in 1922, and today the field is a marginally economic stripper field with average production of less than 3 BOPD (0.5 m{sup 3}/D) per well. Total field production is about 1,800 BOPD (286 m{sup 3}/D). The Second Wall Creek Formation was waterflooded from 1979 until June 1992 with poor results due to the extensive natural fracture system in this sandstone unit. Since water injection ceased, reservoir pressure has declined to very low levels. Liquids extraction and reinjection of the gas produced from high-GOR wells along the gas-oil contact continues, but the average gas cap pressure has fallen to approximately 150 psi (1.03 MPa) from an original pressure of 1,120 psi (7.72 MPa). Since the oil is highly paraffinic, wax deposition in the hydraulic fractures and the perforations has become a serious production problem. Microbial treatment was considered as a possible low-cost solution. Four wells were selected in the Second Wall Creek Reservoir with severe paraffin problems and production rates high enough to economically justify the treatment. Problems were experienced with the production of thick oil after approximately three months. This was interpreted to be a result of previously immobile paraffin being cleaned up. A slight decrease in the decline rate was seen in the wells, although some external factors cloud the interpretation. Microbial treatments were discontinued because of marginal economics. Three of the four wells produced additional oil and had a positive incremental cash flow. Oil viscosity tests did indicate that some positive microbial thinning was occurring, and changes to the treatment procedure may potentially yield more economic results in the future.

Short- and medium-chain chlorinated paraffins are potential PBT chemicals (persistent, bioaccumulative, toxic) and short-chain chlorinated paraffins are under review for inclusion in the UNEP Stockholm Convention on Persistent Organic Pollutants. Despite their high production volume of more than one million metric tonnes per year, only few data on their physicochemical properties are available. We calculated subcooled-liquid vapor pressure, subcooled-liquid solubility in water and octanol, Henry's law constant for water and octanol, as well as the octanol-water partition coefficient with the property calculation methods COSMOtherm, SPARC, and EPI Suite™, and compared the results to experimental data from the literature. For all properties, good or very good agreement between calculated and measured data was obtained for COSMOtherm; results from SPARC were in good agreement with the measured data except for subcooled-liquid water solubility, whereas EPI Suite™ showed the largest discrepancies for all properties. After critical evaluation of the three property calculation methods, a final set of recommended property data for short- and medium-chain chlorinated paraffins was derived. The calculated property data show interesting relationships with chlorine content and carbon chain length. Increasing chlorine content does not cause pronounced changes in water solubility and octanol-water partition coefficient (KOW) as long as it is below 55%. Increasing carbon chain length leads to strong increases in KOW and corresponding decreases in subcooled-liquid water solubility. The present data set can be used in further studies to assess the environmental fate and human exposure of this relevant compound class.

DNA and RNA have been used as markers of tissue quality and integrity throughout the last few decades. In this research study, genomic quality DNA of kidney, liver, heart, lung, spleen, and brain were analyzed in tissues from post-mortem patients and surgical cancer cases spanning the past century. DNA extraction was performed on over 180 samples from: 70+ year old formalin-fixed celloidin-embedded (FFCE) tissues, formalin-fixed paraffin-embedded (FFPE) tissue samples from surgical cases and post-mortem cases from the 1970’s, 1980’s, 1990’s, and 2000’s, tissues fixed in 10% neutral buffered formalin/stored in 70% ethanol from the 1990’s, 70+ year old tissues fixed in unbuffered formalin of various concentrations, and fresh tissue as a control. To extract DNA from FFCE samples and ethanol-soaked samples, a modified standard operating procedure was used in which all tissues were homogenized, digested with a proteinase K solution for a long period of time (24-48 hours), and DNA was extracted using the Autogen Flexstar automated extraction machine. To extract DNA from FFPE, all tissues were soaked in xylene to remove the paraffin from the tissue prior to digestion, and FFPE tissues were not homogenized. The results were as follows: celloidin-embedded and paraffin-embedded tissues yielded the highest DNA concentration and greatest DNA quality, while the formalin in various concentrations, and long term formalin/ethanol-stored tissue yielded both the lowest DNA concentration and quality of the tissues tested. The average DNA yield for the various fixatives was: 367.77 μg/ mL FFCE, 590.7 μg/mL FFPE, 53.74 μg/mL formalin-fixed/70% ethanol-stored and 33.2 μg/mL unbuffered formalin tissues. The average OD readings for FFCE, FFPE, formalin-fixed/70% ethanol-stored tissues, and tissues fixed in unbuffered formalin were 1.86, 1.87, 1.43, and 1.48 respectively. The results show that usable DNA can be extracted from tissue fixed in formalin and embedded in celloidin

An empirical elastohydrodynamic film thickness formula for heavily loaded contacts based upon X-ray film thickness measurements made with a synthetic paraffinic oil is presented. The deduced relation was found to adequately reflect the high load dependence exhibited by the measured minimum film thickness data at high Hertizian contact stresses, that is, above 1.04 x 10 to the ninth N/sq m (150,000 psi). Comparisons were made with the numerical results from a theoretical isothermal film thickness formula. The effects of changes in contact geometry, material, and lubricant properties on the form of the empirical model are also discussed.

We study resonant nonlinear magneto-optic rotation (NMOR) in a paraffin-coated Rb vapor cell as the magnetic field is swept. At low sweep rates, the nonlinear rotation appears as a narrow resonance signal with a linewidth of about “300 μG” (2π×420 Hz). At high sweep rates, the signal shows transient response with an oscillatory decay. The decay time constant is of order 100 ms. The behavior is different for transitions starting from the lower or the upper hyperfine level of the ground state because of optical pumping effects.

Density and kinematic viscosity have been measured with a vibrating tube densimeter and an Ubbelohde capillary viscometer for binary mixtures of 1,1,1-trichloroethane and six paraffins, hexane, heptane, octane, decane, dodecane, and hexadecane, and five cycloparaffins, cyclohexane, methylcyclohexane, ethylcyclohexane, propylcyclohexane, and butylcyclohexane, at atmospheric pressure and 298.15 K. The dynamic viscosity, excess volume, and viscosity deviation function have been obtained from the density and kinematic viscosity results. The results have been correlated with a polynomial expression for the excess volume and with a recently published model for the mixture viscosity. Parameters for such models are reported.

In this article, SnO2nanowires (NWs) have been prepared and their microwave absorption properties have been investigated in detail. Complex permittivity and permeability of the SnO2NWs/paraffin composites have been measured in a frequency range of 0.1-18 GHz, and the measured results are compared with that calculated from effective medium theory. The value of maximum reflection loss for the composites with 20 vol.% SnO2NWs is approximately -32.5 dB at 14 GHz with a thickness of 5.0 mm. PMID:20651925

In this article, SnO2nanowires (NWs) have been prepared and their microwave absorption properties have been investigated in detail. Complex permittivity and permeability of the SnO2NWs/paraffin composites have been measured in a frequency range of 0.1–18 GHz, and the measured results are compared with that calculated from effective medium theory. The value of maximum reflection loss for the composites with 20 vol.% SnO2NWs is approximately −32.5 dB at 14 GHz with a thickness of 5.0 mm. PMID:20651925

Light shifts are an important source of noise and systematics in optically pumped magnetometers. We demonstrate that the long spin coherence time in paraffin-coated cells leads to spatial averaging of the light shifts over the entire cell volume. This renders the averaged light shift independent, under certain approximations, of the light-intensity distribution within the sensor cell. These results and the underlying mechanism can be extended to other spatially varying phenomena in anti-relaxation-coated cells with long coherence times.

Light shifts are an important source of noise and systematics in optically pumped magnetometers. We demonstrate that the long spin-coherence time in paraffin-coated cells leads to spatial averaging of the vector light shift over the entire cell volume. This renders the averaged vector light shift independent, under certain approximations, of the light-intensity distribution within the sensor cell. Importantly, the demonstrated averaging mechanism can be extended to other spatially varying phenomena in anti-relaxation-coated cells with long coherence times. PMID:27410814

Hybrid propulsion technology for aerospace applications has received growing attention in recent years due to its important advantages over competitive solutions. Hybrid rocket engines have a great potential for several aeronautics and aerospace applications because of their safety, reliability, low cost and high performance. As a consequence, this propulsion technology is feasible for a number of innovative missions, including space tourism. On the other hand, hybrid rocket propulsion's main drawback, i.e. the difficulty in reaching high regression rate values using standard fuels, has so far limited the maturity level of this technology. The complex physico-chemical processes involved in hybrid rocket engines combustion are of major importance for engine performance prediction and control. Therefore, further investigation is ongoing in order to achieve a more complete understanding of such phenomena. It is well known that one of the most promising solutions for overcoming hybrid rocket engines performance limits is the use of liquefying fuels. Such fuels can lead to notably increased solid fuel regression rate due to the so-called "entrainment phenomenon". Among liquefying fuels, paraffin-based formulations have great potentials as solid fuels due to their low cost, availability (as they can be derived from industrial waste), low environmental impact and high performance. Despite the vast amount of literature available on this subject, a precise focus on market potential of paraffins for hybrid propulsion aerospace applications is lacking. In this work a review of hybrid rocket engines state of the art was performed, together with a detailed analysis of the possible applications of such a technology. A market study was carried out in order to define the near-future foreseeable development needs for hybrid technology application to the aforementioned missions. Paraffin-based fuels are taken into account as the most promising segment for market development

Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. This protocol describes the fixation and processing required to prepare tissues for immunofluorescence array tomography. PMID:21041396

Biodiesels are promoted as alternative fuels and their applications in diesel engines have been investigated by many researchers. However, the particle size distribution emitted from heavy-duty diesel engines fueled with palm-biodiesel blended with premium diesel fuel and paraffinic fuel blended with palm-biodiesel has seldom been addressed. Thus, five test fuels were used in this work to study the particle size distribution: D100 (premium diesel fuel), B100 (100% palm-biodiesel), B20 (20 vol% palm-biodiesel+80 vol% D100), BP9505 (95 vol% paraffinic fuel+5 vol% palm-biodiesel) and BP8020 (80 vol% paraffinic fuel+20 vol% palm-biodiesel). A Micro-Orifice Uniform Deposit Impactor (MOUDI) equipped with aluminum filters was used to collect size-resolved samples. Experimental results indicated that palm-biodiesel blends and paraffinic fuel blends could improve combustion efficiency in diesel engines, but pure palm-biodiesel could cause incomplete combustion. Adding palm-biodiesel to diesel fuel would slightly increase particles with diameter <0.31 μm but paraffinic fuel blends could decrease particles with diameter <1 μm. The mass median diameter of overall particles (MMD o) and σg,o are 0.439 μm and 3.88 for D100; 0.380 μm and 3.24 for B20; 0.465 μm and 4.22 for B100; 1.40 μm and 4.92 for BP9505; 1.46 μm and 2.25 for BP8020. There are more particles with low aerodynamic diameters (diameter <0.31 μm) in the exhaust of D100, B20 and B100 fuels. On the other hand, a greater fraction of particulate matter of BP9505 and BP8020 existed in coarse particles (diameter: 2.5-10 μm). Energy efficiency also increases significantly by 12.3-15.1% with the introduction of paraffinic fuel blends into the engine. Nevertheless, paraffinic fuel blends also reduce the emission of particulate matters by 36.0-38.4%. Carbon monoxide was decreased by 36.8-48.5%. Total hydrocarbon is 39.6-41.7% less than diesel fuel combustion. Nitrogen oxides emission is about 5% lower for paraffinic

Accurate estimates of scatterer parameters (size and acoustic concentration) are beneficial adjuncts to characterize disease from ultrasonic backscatterer measurements. An estimation technique was developed to obtain parameter estimates from the Fourier transform of the spatial autocorrelation function (SAF). A 3D impedance map (3DZM) is used to obtain the SAF of tissue. 3DZMs are obtained by aligning digitized light microscope images from histologic preparations of tissue. Estimates were obtained for simulated 3DZMs containing spherical scatterers randomly located: relative errors were less than 3%. Estimates were also obtained from a rat fibroadenoma and a 4T1 mouse mammary tumor (MMT). Tissues were fixed (10% neutral-buffered formalin), embedded in paraffin, serially sectioned and stained with H&E. 3DZM results were compared to estimates obtained independently against ultrasonic backscatter measurements. For the fibroadenoma and MMT, average scatterer diameters were 91 and 31.5 μm, respectively. Ultrasonic measurements yielded average scatterer diameters of 105 and 30 μm, respectively. The 3DZM estimation scheme showed results similar to those obtained by the independent ultrasonic measurements. The 3D impedance maps show promise as a powerful tool to characterize ultrasonic scattering sites of tissue. [Work supported by the University of Illinois Research Board.

We generalize Israel's formalism to cover singular shells embedded in a non-vacuum Universe. That is, we deduce the relativistic equation of motion for a thin shell embedded in a Schwarzschild/Friedmann-Lemaître-Robertson-Walker spacetime. Also, we review the embedding of a Schwarzschild mass into a cosmological model using ``curvature'' coordinates and give solutions with (Sch/FLRW) and without the embedded mass (FLRW).

Recent modifications have been made to generalize the Embedded Atom Method (EAM) to describe bonding in diverse materials. By including angular dependence of the electron density in an empirical way, the Modified Embedded Atom Method (MEAM) has been able to reproduce the basic energetic and structural properties of 45 elements. This method is ideally suited for examining the interfacial behavior of dissimilar materials. This paper explains in detail the derivation of the method, shows how the parameters of the MEAM are determined directly from experiment or first principles calculations, and examines the quality of the reproduction of the database. Materials with fcc, bcc, hcp, and diamond cubic crystal structure are discussed. A few simple examples of the application of the MEAM to surfaces and interfaces are presented. Calculations of pullout of a SiC fiber in a diamond matrix as a function of applied stress show non-uniform deformation of the fiber.

A Miniature Embedded Reconfigurable Computer and Logic (MERCAL) module has been developed and verified. MERCAL was designed to be a general-purpose, universal module that that can provide significant hardware and software resources to meet the requirements of many of today's complex embedded applications. This is accomplished in the MERCAL module by combining a sub credit card size PC in a DIMM form factor with a XILINX Spartan I1 FPGA. The PC has the ability to download program files to the FPGA to configure it for different hardware functions and to transfer data to and from the FPGA via the PC's ISA bus during run time. The MERCAL module combines, in a compact package, the computational power of a 133 MHz PC with up to 150,000 gate equivalents of digital logic that can be reconfigured by software. The general architecture and functionality of the MERCAL hardware and system software are described.

This progress report describes research towards the design and construction of embedded operating systems for real-time advanced aerospace applications. The applications concerned require reliable operating system support that must accommodate networks of computers. The report addresses the problems of constructing such operating systems, the communications media, reconfiguration, consistency and recovery in a distributed system, and the issues of realtime processing. A discussion is included on suitable theoretical foundations for the use of atomic actions to support fault tolerance and data consistency in real-time object-based systems. In particular, this report addresses: atomic actions, fault tolerance, operating system structure, program development, reliability and availability, and networking issues. This document reports the status of various experiments designed and conducted to investigate embedded operating system design issues.

This paper presents an experimental study of the implementation of a face recognition system in embedded systems. To investigate the feasibility and practicality of real time face recognition on such systems, a door access control system based on face recognition is built. Due to the limited computation power of embedded device, a semi-automatic scheme for face detection and eye location is proposed to solve these computationally hard problems. It is found that to achieve real time performance, optimization of the core face recognition module is needed. As a result, extensive profiling is done to pinpoint the execution hotspots in the system and optimization are carried out. After careful precision analysis, all slow floating point calculations are replaced with their fixed-point versions. Experimental results show that real time performance can be achieved without significant loss in recognition accuracy.

The long term durability of WAXFIXi, a paraffin based grout, was evaluated for in situ grouting of activated beryllium wastes in the Subsurface Disposal Area (SDA), a radioactive landfill at the Radioactive Waste Management Complex, part of the Idaho National Laboratory (INL). The evaluation considered radiological and biological mechanisms that could degrade the grout using data from an extensive literature search and previous tests of in situ grouting at the INL. Conservative radioactive doses for WAXFIX were calculated from the "hottest" (i.e., highest-activity) Advanced Test Reactor beryllium block in the SDA.. These results indicate that WAXFIX would not experience extensive radiation damage for many hundreds of years. Calculation of radiation induced hydrogen generation in WAXFIX indicated that grout physical performance should not be reduced beyond the effects of radiation dose on the molecular structure. Degradation of a paraffin-based grout by microorganisms in the SDA is possible and perhaps likely, but the rate of degradation will be at a slower rate than found in the literature reviewed. The calculations showed the outer 0.46 m (18 in.) layer of each monolith, which represents the minimum expected distance to the beryllium block, was calculated to require 1,000 to 3,600 years to be consumed. The existing data and estimations of biodegradation and radiolysis rates

The following paper describes a six month pilot test for the control of paraffin and asphaltene deposition at the reservoir level using naturally occurring microorganisms. For the course of the paper, paraffin will be defined as pentane soluble hydrocarbon precipitates. Likewise, asphaltene will be defined as hydrocarbon deposits which are pentane insoluble and toluene soluble. The microbial project was conducted on four wells in the Eastern Operating Area of the Prudhoe Bay oil field. The microorganisms were selected based upon their ability to metabolize long hydrocarbon chains into lighter components. Data collected from the project reflects the impact on total fluid production and crude oil physical and chemical properties i.e. API gravity, viscosity, and sulfur content. Operation and maintenance costs for the microbial treatments versus alternative methods are shown and proven to be favorable on certain well types. The advantages of growing the microbes on site and increasing the variety and type of microbial functions is presented. The treatment techniques for effective microbial application along with candidate selection are discussed.

We report on a patient who was diagnosed with high-grade breast carcinoma by all the pre-surgery clinical evidence of malignancy, but histopathological reports did not reveal any such tumor residue in the post-surgical tissue block. This raised a suspicion that either exchange of block, labeling error, or a technical error took place during gross examination of the tissue. The mastectomy residue was unprocurable to sort out the problem. So, two doubtful paraffin blocks were sent for DNA fingerprinting analysis. The partial DNA profiles (8-9/15 loci) were obtained from histocytological blocks. The random matching probability for both the paraffin blocks and the patient’s blood were found to be 1 in 4.43E4, 1.89E6, and 8.83E13, respectively for Asian population. Multiplex short tandem repeat analysis applied in this case determined that the cause of tumor absence was an error in gross examination of the post-surgical tissue. Moreover, the analysis helped in justifying the therapy given to the patient. Thus, with DNA fingerprinting technique, it was concluded that there was no exchange of the blocks between the two patients operated on the same day and the treatment given to the concerned patient was in the right direction. PMID:21674839

Embedded librarianship service describes the practice of librarians integrating actively into the user's environment, rather than remaining in the library to await requests for service. This paper examines the concept of embedded service in the recent literature, within the past 5 years. It reports on a survey of embedded service in Chinese…

All known methods of lossless or reversible data embedding that exist today suffer from two major disadvantages: 1) The embedded image suffers from distortion, however small it may be by the very process of embedding and 2) The requirement of a special parser (decoder), which is necessary for the client to remove the embedded data in order to obtain the original image (lossless). We propose a novel lossless data embedding method where both these disadvantages are circumvented. Zero-distortion lossless data embedding (ZeroD-LDE) claims 'zero-distortion' of the embedded image for all viewing purposes and further maintaining that clients without any specialized parser can still recover the original image losslessly but would not have direct access to the embedded data. The fact that not all gray levels are used by most images is exploited to embed data by selective lossless compression of run-lengths of zeros (or any compressible pattern). Contiguous runs of zeros are changed such that the leading zero is made equal to the maximum original intensity plus the run-length and the succeeding zeros are converted to the embedded data (plus maximum original intensity) thus achieving extremely high embedding capacities. This way, the histograms of the host-data and the embedded data do not overlap and hence we can obtain zero-distortion by using the window-level setting of standard DICOM viewers. The embedded image is thus not only DICOM compatible but also zero-distortion visually and requires no clinical validation.

We study an autonomous four-dimensional dynamical system used to model certain geophysical processes. This system generates a chaotic attractor that is strongly contracting, with four Lyapunov exponents λi that satisfy λ1+λ2+λ3<0 , so the Lyapunov dimension is DL=2+∣λ3∣/λ1<3 in the range of coupling parameter values studied. As a result, it should be possible to find three-dimensional spaces in which the attractors can be embedded so that topological analyses can be carried out to determine which stretching and squeezing mechanisms generate chaotic behavior. We study mappings into R3 to determine which can be used as embeddings to reconstruct the dynamics. We find dramatically different behavior in the two simplest mappings: projections from R4 to R3 . In one case the one-parameter family of attractors studied remains topologically unchanged for all coupling parameter values. In the other case, during an intermediate range of parameter values the projection undergoes self-intersections, while the embedded attractors at the two ends of this range are topologically mirror images of each other.

We study an autonomous four-dimensional dynamical system used to model certain geophysical processes. This system generates a chaotic attractor that is strongly contracting, with four Lyapunov exponents lambdai that satisfy lambda1+lambda2+lambda3<0 , so the Lyapunov dimension is DL=2+|lambda3|/lambda1<3 in the range of coupling parameter values studied. As a result, it should be possible to find three-dimensional spaces in which the attractors can be embedded so that topological analyses can be carried out to determine which stretching and squeezing mechanisms generate chaotic behavior. We study mappings into R3 to determine which can be used as embeddings to reconstruct the dynamics. We find dramatically different behavior in the two simplest mappings: projections from R4 to R3 . In one case the one-parameter family of attractors studied remains topologically unchanged for all coupling parameter values. In the other case, during an intermediate range of parameter values the projection undergoes self-intersections, while the embedded attractors at the two ends of this range are topologically mirror images of each other. PMID:17500972

In the article density-orbital embedding (DOE) theory is proposed. DOE is based on the concept of density orbital (DO), which is a generalization of the square root of the density for real functions and fractional electron numbers. The basic feature of DOE is the representation of the total supermolecular density {rho}{sub s} as the square of the sum of the DO {phi}{sub a}, which represents the active subsystem A and the square root of the frozen density {rho}{sub f} of the environment F. The correct {rho}{sub s} is obtained with {phi}{sub a} being negative in the regions in which {rho}{sub f} might exceed {rho}{sub s}. This makes it possible to obtain the correct {rho}{sub s} with a broad range of the input frozen densities {rho}{sub f} so that DOE resolves the problem of the frozen-density admissibility of the current frozen-density embedding theory. The DOE Euler equation for the DO {phi}{sub a} is derived with the characteristic embedding potential representing the effect of the environment. The DO square {phi}{sub a}{sup 2} is determined from the orbitals of the effective Kohn-Sham (KS) system. Self-consistent solution of the corresponding one-electron KS equations yields not only {phi}{sub a}{sup 2}, but also the DO {phi}{sub a} itself.

The dimension of a system is one of the most fundamental quantities to characterize its structure and basic physical properties. Diffusion and vibrational excitations, for example, as well as the universal features of a system near a critical point depend crucially on its dimension. However, in the theory of complex networks the concept of dimension has been rarely discussed. Here we study models for spatially embedded networks and show how their dimension can be determined. Our results indicate that networks characterized by a broad distribution of link lengths have a dimension higher than that of the embedding space. We illustrate our findings using the global airline network and the Internet and argue that although these networks are embedded in two-dimensional space they should be regarded as systems with dimension close to 3 and 4.5, respectively. We show that the network dimension is a key concept to understand not only network topology, but also dynamical processes on networks, such as diffusion and critical phenomena including percolation.

Many optimization problems are defined on highly connected graphs and many interesting physical spin-glass systems are featuring long-range interactions. One method to solve for the optimum/ground state is quantum annealing (QA). Most architectures for QA devices, manufactured or proposed, are based on optimizing Hamiltonians having spins connected in a non-complete graph, with nodes with a small maximum degree, compared to the requirements. To overcome this limitation 'embedding' is employed: the native graph is 'tiled' with ferromagnetic chains of spins that now are meant to represent the logical binary variables. While it is known how the strength of the ferromagnetic bonds can ensure that the classical Ising ground state of the embedded system can be univocally mapped to the ground state of the original system, there is very little study on the impact of these parameters on QA. Programmers have taken conservative choices for the parameters and the common practices can be improved. Starting from the physics of connected ferromagnetic Ising chains, we will review several parameter choices and discuss previous and new results obtained on the D-Wave 2X machine, on carefully designed problems that allow to isolate and evaluate the role of connectivity in embedded systems.

The MLA and IFA of the instrument on the IceCube require a 20 C temperature and a thermal stability of +/-1 C. The thermal environment of the ISS orbit for the IceCube is very unstable due to solar beta angles in the -75deg to +75deg range. Additionally the instrument is powered off in every eclipse to conserve electrical power. These two factors cause thermal instability to the MLA and IFA. This paper presents a thermal design of using mini paraffin PCM packs to meet the thermal requirements of these instrument components. With a 31 g mass plus a 30% margin of n-hexadecane, the MLA and IFA are powered on for 32.3 minutes in sunlight at a 0deg beta angle to melt the paraffin. The powered-on time increases to 38 minutes at a 75deg (+/-) beta angle. When the MLA and IFA are powered off, the paraffin freezes.

In the framework of a long-term research activity focused on the development of high-performance solid fuels for hybrid rockets, paraffin-based fuels were investigated and characterized using two different pure paraffinic waxes and a styrene-based thermoplastic elastomer as strengthening material. The fuels were studied using differential scanning calorimetry (DSC) and thermogravimetric analysis / differential thermal analysis (TGA-DTA). The viscosity of the melt layer, responsible for the entrainment effect, was investigated using a Couette viscosimeter. The storage modulus (G') was analyzed using a parallel-plate rheometer. The chemical composition of the pure paraffinic materials was studied using gas chromatography / mass spectrometry (GC-MS), while mechanical properties were investigated through uniaxial tensile tests.

In 1998, paraffin tissue microarrays (PTMAs) as paraffin blocks containing up to 1,000 cylindrical paraffin tissue core biopsy specimens (PTCBs) for high-throughput molecular profiling of tumor specimens were introduced. PTCBs can be constructed using a manual tissue puncher/arrayer (Beecher Instruments, Sun Prairie, WI; cost, at least $7,000). Furthermore, custom-built PTMAs such as the MaxArray are created by companies such as Zymed Laboratories (South San Francisco, CA; PTMA with 96 holes, about $900). In our search for a less expensive alternative, we constructed PTMAs with up to 558 PTCBs by using a drill grinder, a drill stand, and a microcompound table (Proxxon, Niersdorf, Germany; cost,

In contrast to imaging modalities such as magnetic resonance imaging and micro computed tomography, digital histology reveals multiple stained tissue features at high resolution (0.25μm/pixel). However, the two-dimensional (2D) nature of histology challenges three-dimensional (3D) quantification and visualization of the different tissue components, cellular structures, and subcellular elements. This limitation is particularly relevant to the vasculature, which has a complex and variable structure within tissues. The objective of this study was to perform a fully automated 3D reconstruction of histology tissue in the mouse hind limb preserving the accurate systemic orientation of the tissues, stained with hematoxylin and immunostained for smooth muscle α actin. We performed a 3D reconstruction using pairwise rigid registrations of 5μm thick, paraffin-embedded serial sections, digitized at 0.25μm/pixel. Each registration was performed using the iterative closest points algorithm on blood vessel landmarks. Landmarks were vessel centroids, determined according to a signed distance map of each pixel to a decision boundary in hue-saturation-value color space; this decision boundary was determined based on manual annotation of a separate training set. Cell nuclei were then automatically extracted and corresponded to refine the vessel landmark registration. Homologous nucleus landmark pairs appearing on not more than two adjacent slides were chosen to avoid registrations which force curved or non-sectionorthogonal structures to be straight and section-orthogonal. The median accumulated target registration errors ± interquartile ranges for the vessel landmark registration, and the nucleus landmark refinement were 43.4+/-42.8μm and 2.9+/-1.7μm, respectively (p<0.0001). Fully automatic and accurate 3D rigid reconstruction of mouse hind limb histology imaging is feasible based on extracted vasculature and nuclei.

In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize in situ probes in thin or ultrathin paraffinembedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or "tree" in situ method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections. PMID:25794171

This study investigated the genetic features of Toxoplasma gondii isolated directly in autopsies of HIV-infected patients who died with severe disseminated toxoplasmosis. This retrospective analysis was conducted in a cohort of 15 HIV-infected patients with clinical and laboratory data. They had previous cerebral toxoplasmosis at least 6 months before the disseminated toxoplasmosis episode. The hypothesis was that they were infected with highly virulent parasites due to the condition in which they died. T. gondii genotyping was done directly in DNA extracted from 30 autopsy brain and lung samples (2 per patient) and mutilocus PCR-RFLP genotyping was done using 12 molecular markers. The 30 clinical samples were genotyped successfully in 8 or more loci and six suggestive genotypes were identified. One of them was Toxo DB #11, previously identified in different domestic animals and virulent in experimental animals. The other five suggestive genotypes identified in 14 patients were not described. TgHuDis1 was the most frequent and was determined in 8 patients. TgHuDis3 and TgHuDis5 were identified in two patients each. TgHuDis2 and TgHuDis4 have been identified in one patient each. These suggestive genotypes could be considered as virulent, since they caused severe tissue damage and had similar characteristics as Toxo # DB 11. PMID:26969767

Transmissible spongiform encephalopathies including bovine spongiform encephalopathy and scrapie are fatal neurodegenerative disorders associated with the presence of an infectious abnormal isoform of normal mammalian proteins called prions (PrP**Sc). Identification of PrP**Sc in the CNS is typicall...

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples. PMID:26484710

Reticuloendotheliosis virus (REV) infection can result in immunosuppression, runting syndrome, high mortality, acute reticulum cell neoplasia, or T- and/or B-cell lymphomas, in a variety of domestic and wild birds. Histopathological changes in REV infection are not sufficient to differentiate it fro...

A Cesarean section (C-section) is surgery to deliver a baby. The baby is taken out through the mother's abdomen. In the United ... three women has their babies this way. Some C-sections are planned, but many are done when ...

A Cesarean section (C-section) is surgery to deliver a baby. The baby is taken out through the mother's abdomen. In the United ... four women have their babies this way. Most C-sections are done when unexpected problems happen during ...

Comprehensive survey is given of the thermal aspects of phase change material devices. Fundamental mechanisms of heat transfer within the phase change device are discussed. Performance in zero-g and one-g fields are examined as it relates to such a device. Computer models for phase change materials, with metal fillers, undergoing conductive and convective processes are detailed. Using these models, extensive parametric data are presented for a hypothetical configuration with a rectangular phase change housing, using straight fins as the filler, and paraffin as the phase change material. These data are generated over a range of realistic sizes, material properties, and thermal boundary conditions. A number of illustrative examples are given to demonstrate use of the parametric data. Also, a complete listing of phase change material property data are reproduced herein as an aid to the reader.

This document describes the results of a DOE funded joint effort of Membrane Technology and Research Inc. (MTR), SRI International (SRI), and ABB Lummus (ABB) to develop facilitated transport membranes for olefin/paraffin separations. Currently, olefin/paraffin separation is done by distillation—an extremely energy-intensive process because of the low relative volatilities of olefins and paraffins. If facilitated transport membranes could be successfully commercialized, the potential energy savings achievable with this membrane technology are estimated to be 48 trillion Btu per year by the year 2020. We discovered in this work that silver salt-based facilitated transport membranes are not stable even in the presence of ideal olefin/paraffin mixtures. This decline in membrane performance appears to be caused by a previously unrecognized phenomenon that we have named olefin conditioning. As the name implies, this mechanism of performance degradation becomes operative once a membrane starts permeating olefins. This project is the first study to identify olefin conditioning as a significant factor impacting the performance of facilitated olefin transport membranes. To date, we have not identified an effective strategy to mitigate the impact of olefin conditioning. other than running at low temperatures or with low olefin feed pressures. In our opinion, this issue must be addressed before further development of facilitated olefin transport membranes can proceed. In addition to olefin conditioning, traditional carrier poisoning challenges must also be overcome. Light, hydrogen, hydrogen sulfide, and acetylene exposure adversely affect membrane performance through unwanted reaction with silver ions. Harsh poisoning tests with these species showed useful membrane lifetimes of only one week. These tests demonstrate a need to improve the stability of the olefin complexing agent to develop membranes with lifetimes satisfactory for commercial application. A successful effort

In this study, nanoscale zero-valent iron (NZVI) particles were synthesized and used for the reductive dehalogenation of short chain chlorinated paraffins (SCCPs) in the laboratory. The results show that the dechlorination rate of chlorinated n-decane (CP(10)) by NZVI increased with decreased solution pH. Increasing the loading of NZVI enhanced the dechlorination rate of CP(10). With an increase in temperature, the degradation rate increased. The reduction of CP(10) by NZVI was accelerated with increasing the concentration of humic acid up to 15 mg/L but then was inhibited. The dechlorination of CP(10) within the initial 18 h followed pseudo-first order rate model. The formation of intermediate products indicates a stepwise dechlorination pathway of SCCPs by NZVI. The carbon chain length and chlorination degree of SCCPs have a polynominal impact on dechlorination reactions. PMID:23107289

Formation of gasoline and diesel fuel has been investigated using three various radiation-induced ways: (1) cracking of wax, (2) synthesis from methane, (3) high-temperature conversion of wax dilute solution in methane. The wax, synthesized by Fischer-Tropsch method, initially contained a mixture of C17-C120 linear paraffins. The yield of wax conversion to liquid mixture (C4-C27 alkenes and 61.5% alkanes) via mode (1) was 0.83±0.09 μmole/J, whereas yield of gas conversion to liquid mixture (C5-C13 alkanes) via mode (2) was 0.95±0.02 μmole/J. In the dilute solution wax underwent indirect action of radiation. In comparison with (1) the mode (3) produces similar amount of lighter fuel containing 80% of alkanes (C5-C15). At the same time degree of methane fixation is almost three times higher.

The ordered mesoporous carbon (OMC)/paraffin composites were successfully prepared by a facile physical mixing method and an EMI SE of 21-23 dB was achieved at the OMC loading of 5.69 wt.% in the X band. This indicates that the composites are very suitable for an application as effective and lightweight EMI shielding materials. The EMI shielding of the composite shows an absorption-dominant mechanism, i.e., a contribution shift from reflection to absorption is observed with the increase in OMC loading and frequency. This could be explained by the intrinsic properties (electrical conductivity, complex permittivity and potential large defects) and novel structure of the composites. PMID:25936048

In regions with a humid summer climate, the use of water-soluble bait to control apple maggot is often limited by rainfall. We studied increasing the rainfastness of GF-120 fruit fly bait by adding paraffin wax emulsion. First, we verified that adding 10% wax to a mixture containing 16.7% GF-120 did not reduce the mortality of female apple maggot compared with GF-120 without wax. In addition, we determined that fly mortality caused by GF-120 plus wax subjected to simulated rain was similar to that caused by GF-120 without wax not subjected to rain. Other assays showed that higher fly mortality resulted from increasing the proportion of wax from 10 to 15%, and lower mortality resulted from decreasing GF-120 from 16.7 to 10 or 5%. The availability of spinosad on or near droplets of a mixture consisting of 5, 10, or 15% GF-120 and 15% wax was determined before and after the droplets were subjected to three 15-min periods of simulated rain. We found an initial steep decline in dislodgeable spinosad and smaller decreases after subsequent periods of rain. In a small-plot field trial, fruit infestation by apple maggot in plots treated with a mixture consisting of 16.7% GF-120 and 19.2% wax was less than in plots treated with 16.7% GF-120 without wax but not less than in control plots. Overall, we found that adding paraffin wax emulsion to GF-120 increased rainfastness in laboratory bioassays, and specifically that it retained the active ingredient spinosad. However, our field data suggest that optimal rainfastness requires the development of mixtures with > 19.2% wax, which may preclude application using standard spray equipment. PMID:19610426

Experiments were conducted in 0.05 m ID and 0.23 m ID by 3 m tall bubble columns with different types of molten waxes as the liquid medium and nitrogen as the gas, under processing conditions typical or Fischer-Tropsch synthesis over iron catalysts (i.e. gas velocities up to 0.15 m s, and temperatures between 200 and 270/sup 0/C) to estimate gas liquid interfacial area from measured values of average gas hold-up and Sauter mean bubble diameter. The gas hold-up was estimated from visual observations of the expanded and static liquid heights, and the Sauter was estimated from bubble size measurements obtained by photography and dynamic gas disengagement. The paraffin wax (FT-300) used in the authors' studies is non-coalescing and has a tendency to foam. The amount of foam is greater for runs conducted in the order of increasing gas velocities, than in runs with decreasing velocities. Thus, two values of hold-up are possible and the start-up procedure determines which one will be attained. At higher gas velocities (> 0.05 m/s) the foam disappears and a transition to the slug flow, churn-turbulent regime takes place. Reactor waxes are coalescing in nature and do not produce foam. Despite similar hold-ups for the different waxes at higher gas velocities, the Sauters are significantly different and this is reflected in the specific gas-liquid interfacial areas, with larger values obtained with the paraffin wax compared to values with reactor waxes.

Embedding a new programming language into an existing one is a widely used technique, because it fastens the development process and gives a part of a language infrastructure for free (e.g. lexical, syntactical analyzers). In this paper we are presenting a new advantage of this development approach regarding to adding testing support for these new languages. Tool support for testing is a crucial point for a newly designed programming language. It could be done in the hard way by creating a testing tool from scratch, or we could try to reuse existing testing tools by extending them with an interface to our new language. The second approach requires less work, and also it fits very well for the embedded approach. The problem is that the creation of such interfaces is not straightforward at all, because the existing testing tools were mostly not designed to be extendable and to be able to deal with new languages. This paper presents an extendable and modular model of a testing framework, in which the most basic design decision was to keep the - previously mentioned - interface creation simple and straightforward. Other important aspects of our model are the test data generation, the oracle problem and the customizability of the whole testing phase.

Paraffins are naturally-occurring components of crude oils, but often form solids within oil reservoirs and on oil production equipment when oil is harvested from hot subsurface temperatures to the cooler surface environments. Microbial t...

In a recent paper (Zuo et al., Appl Catal A 408:130-136, 2011), the mechanism of dimethyl ether (DME) synthesis from methanol dehydration over γ-Al2O3 (110) was studied using density functional theory (DFT). Using the same method, the effect of surface hydroxyls on γ-Al2O3 in liquid paraffin during DME synthesis from methanol dehydration is investigated. It is found that DME is mainly formed from two adsorbed CH3O groups via methanol dehydrogenation on both dehydrated and hydrated γ-Al2O3 in liquid paraffin. No close correlation between catalytic activity and acid intensity was found. Before and after water adsorption at typical catalytic conditions (e.g., 553 K), the reaction rate is not obviously changed on γ-Al2O3(100) surface in liquid paraffin, but the reaction rate decreases by about 11 times on the (110) in liquid paraffin. Considering the area of the (110) and (100) surfaces under actual conditions, the catalytic activity decreased mainly because the Al3 sites on the (110) surface gradually become inactive. Catalytic activity decreased mainly due to surface hydrophilicity. The calculated results were consistent with the experiment. PMID:24057976

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffinembedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may

Embedding nanostructures within a bulk matrix is an important practical approach towards the electronic engineering of high performance thermoelectric systems. For power generation applications, it ideally combines the efficiency benefit offered by low dimensional systems along with the high power output advantage offered by bulk systems. In this work, we uncover a few crucial details about how to embed nanowires and nanoflakes in a bulk matrix so that an overall advantage over pure bulk may be achieved. First and foremost, we point out that a performance degradation with respect to bulk is inevitable as the nanostructure transitions to a multi moded one. It is then shown that a nano embedded system of suitable cross-section offers a power density advantage over a wide range of efficiencies at higher packing fractions, and this range gradually narrows down to the high efficiency regime, as the packing fraction is reduced. Finally, we introduce a metric - the advantage factor, to elucidate quantitatively, the enhancement in the power density offered via nano-embedding at a given efficiency. In the end, we explore the maximum effective width of nano-embedding which serves as a reference in designing generators in the efficiency range of interest.

Embedding nanostructures within a bulk matrix is an important practical approach towards the electronic engineering of high performance thermoelectric systems. For power generation applications, it ideally combines the efficiency benefit offered by low dimensional systems along with the high power output advantage offered by bulk systems. In this work, we uncover a few crucial details about how to embed nanowires and nanoflakes in a bulk matrix so that an overall advantage over pure bulk may be achieved. First and foremost, we point out that a performance degradation with respect to bulk is inevitable as the nanostructure transitions to a multi moded one. It is then shown that a nano embedded system of suitable cross-section offers a power density advantage over a wide range of efficiencies at higher packing fractions, and this range gradually narrows down to the high efficiency regime, as the packing fraction is reduced. Finally, we introduce a metric - the advantage factor, to elucidate quantitatively, the enhancement in the power density offered via nano-embedding at a given efficiency. In the end, we explore the maximum effective width of nano-embedding which serves as a reference in designing generators in the efficiency range of interest.

We review two recent developments on five-dimensional embedding theories of the spacetime: the embedding of branes, which is related to the Campbell-Magaard theorem, and the dynamics of particles in the context of the induced-matter theory. In the latter case, we show that there are two different ways of treating the anomalous acceleration of particles: the foliation and the embedding approach.